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
3759,ERR1294279,ERX1365625,ERS1067820,ERP014370,PRJEB12848,Danio rerio Mau mutants skin Transcriptome,ena-STUDY-MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY-25-02-2016-18:42:00:594-265,Other,Mau/Aqp3a dominant mutants of zebrafish Danio rerio are characterized by broken stripes and short fins. To identify signaling pathways affected by mau mutations and leading to pigment patterning defects we analyze skin transcriptome of adult wild type TU fish and two mau alleles.,ENA FIRST PUBLIC:2016 05 24|ENA LAST UPDATE:2016 02 25,,,Mau tVBU1/tVBU1 skin transcriptome,SAMEA3880686,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,ENA first public:2016 05 24|ENA last update:2016 02 25|External Id:SAMEA3880686|INSDC center alias:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC center name:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC first public:2016 05 24T17:02:01Z|INSDC last update:2016 02 25T18:42:06Z|INSDC status:public|Submitter Id:VBU1|allele:mau tVBU1/tVBU1|common name:zebrafish|dev stage:adult|sample name:VBU1|tissue type:skin,,,,,,,,,Illumina HiSeq 2000 sequencing,ena EXPERIMENT MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 25 02 2016 18:41:58:670 9,vbu13,1,Illumina TruSeq RNA,,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2000,,ERP014370,Illumina HiSeq 2000 sequencing,ENA FIRST PUBLIC:2016 05 24|ENA LAST UPDATE:2018 11 16,vbu13_GCCAAT_L008_R1_001.fastq.gz,fastq,2376143675.0,23526175.0,ena RUN MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 25 02 2016 18:41:58:670 9,0:101,A:655940769;C:534990399;G:527934646;T:655773043;N:1504818,101,,,,655940769,534990399,527934646,655773043,1504818,ERX1365625,ERS1067820,ERA567249,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|European Nucleotide Archive,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,1,0.95443,,0.10666,,0.73176,,0.50978,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2016-02-25,Adult,Adult,Skin,Surface Structure
3760,ERR1294278,ERX1365624,ERS1067820,ERP014370,PRJEB12848,Danio rerio Mau mutants skin Transcriptome,ena-STUDY-MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY-25-02-2016-18:42:00:594-265,Other,Mau/Aqp3a dominant mutants of zebrafish Danio rerio are characterized by broken stripes and short fins. To identify signaling pathways affected by mau mutations and leading to pigment patterning defects we analyze skin transcriptome of adult wild type TU fish and two mau alleles.,ENA FIRST PUBLIC:2016 05 24|ENA LAST UPDATE:2016 02 25,,,Mau tVBU1/tVBU1 skin transcriptome,SAMEA3880686,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,ENA first public:2016 05 24|ENA last update:2016 02 25|External Id:SAMEA3880686|INSDC center alias:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC center name:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC first public:2016 05 24T17:02:01Z|INSDC last update:2016 02 25T18:42:06Z|INSDC status:public|Submitter Id:VBU1|allele:mau tVBU1/tVBU1|common name:zebrafish|dev stage:adult|sample name:VBU1|tissue type:skin,,,,,,,,,Illumina HiSeq 2000 sequencing,ena EXPERIMENT MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 25 02 2016 18:41:58:670 8,vbu12,1,Illumina TruSeq RNA,,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2000,,ERP014370,Illumina HiSeq 2000 sequencing,ENA FIRST PUBLIC:2016 05 24|ENA LAST UPDATE:2018 11 16,vbu12_GTGAAA_L008_R1_001.fastq.gz,fastq,1449181835.0,14348335.0,ena RUN MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 25 02 2016 18:41:58:670 8,0:101,A:399518584;C:327615685;G:323868050;T:397240465;N:939051,101,,,,399518584,327615685,323868050,397240465,939051,ERX1365624,ERS1067820,ERA567249,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|European Nucleotide Archive,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,1,0.95893,,0.09315,,0.73464,,0.48744,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2016-02-25,Adult,Adult,Skin,Surface Structure
3761,ERR1294277,ERX1365623,ERS1067820,ERP014370,PRJEB12848,Danio rerio Mau mutants skin Transcriptome,ena-STUDY-MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY-25-02-2016-18:42:00:594-265,Other,Mau/Aqp3a dominant mutants of zebrafish Danio rerio are characterized by broken stripes and short fins. To identify signaling pathways affected by mau mutations and leading to pigment patterning defects we analyze skin transcriptome of adult wild type TU fish and two mau alleles.,ENA FIRST PUBLIC:2016 05 24|ENA LAST UPDATE:2016 02 25,,,Mau tVBU1/tVBU1 skin transcriptome,SAMEA3880686,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,ENA first public:2016 05 24|ENA last update:2016 02 25|External Id:SAMEA3880686|INSDC center alias:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC center name:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC first public:2016 05 24T17:02:01Z|INSDC last update:2016 02 25T18:42:06Z|INSDC status:public|Submitter Id:VBU1|allele:mau tVBU1/tVBU1|common name:zebrafish|dev stage:adult|sample name:VBU1|tissue type:skin,,,,,,,,,Illumina HiSeq 2000 sequencing,ena EXPERIMENT MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 25 02 2016 18:41:58:670 7,vbu11,1,Illumina TruSeq RNA,,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2000,,ERP014370,Illumina HiSeq 2000 sequencing,ENA FIRST PUBLIC:2016 05 24|ENA LAST UPDATE:2018 11 16,vbu11_GTCCGC_L008_R1_001.fastq.gz,fastq,1455235169.0,14408269.0,ena RUN MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 25 02 2016 18:41:58:670 7,0:101,A:392100064;C:337980617;G:331091626;T:393178582;N:884280,101,,,,392100064,337980617,331091626,393178582,884280,ERX1365623,ERS1067820,ERA567249,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|European Nucleotide Archive,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,1,0.95485,,0.08016,,0.72845,,0.47549,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2016-02-25,Adult,Adult,Skin,Surface Structure
3762,ERR1294276,ERX1365622,ERS1067819,ERP014370,PRJEB12848,Danio rerio Mau mutants skin Transcriptome,ena-STUDY-MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY-25-02-2016-18:42:00:594-265,Other,Mau/Aqp3a dominant mutants of zebrafish Danio rerio are characterized by broken stripes and short fins. To identify signaling pathways affected by mau mutations and leading to pigment patterning defects we analyze skin transcriptome of adult wild type TU fish and two mau alleles.,ENA FIRST PUBLIC:2016 05 24|ENA LAST UPDATE:2016 02 25,,,Mau tVE1/tVE1 skin transcriptome,SAMEA3880685,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,ENA first public:2016 05 24|ENA last update:2016 02 25|External Id:SAMEA3880685|INSDC center alias:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC center name:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC first public:2016 05 24T17:02:01Z|INSDC last update:2016 02 25T18:42:06Z|INSDC status:public|Submitter Id:VE1|allele:mau tVE1/tVE1|common name:zebrafish|dev stage:adult|sample name:VE1|tissue type:skin,,,,,,,,,Illumina HiSeq 2000 sequencing,ena EXPERIMENT MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 25 02 2016 18:41:58:670 6,ve13,1,Illumina TruSeq RNA,,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2000,,ERP014370,Illumina HiSeq 2000 sequencing,ENA FIRST PUBLIC:2016 05 24|ENA LAST UPDATE:2018 11 16,ve13_CCGTCC_L008_R1_001.fastq.gz,fastq,1029294030.0,10191030.0,ena RUN MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 25 02 2016 18:41:58:670 6,0:101,A:284612731;C:231451012;G:228443320;T:284135536;N:651431,101,,,,284612731,231451012,228443320,284135536,651431,ERX1365622,ERS1067819,ERA567249,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|European Nucleotide Archive,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,1,0.93287,,0.10726,,0.72585,,0.4913,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2016-02-25,Adult,Adult,Skin,Surface Structure
3763,ERR1294275,ERX1365621,ERS1067819,ERP014370,PRJEB12848,Danio rerio Mau mutants skin Transcriptome,ena-STUDY-MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY-25-02-2016-18:42:00:594-265,Other,Mau/Aqp3a dominant mutants of zebrafish Danio rerio are characterized by broken stripes and short fins. To identify signaling pathways affected by mau mutations and leading to pigment patterning defects we analyze skin transcriptome of adult wild type TU fish and two mau alleles.,ENA FIRST PUBLIC:2016 05 24|ENA LAST UPDATE:2016 02 25,,,Mau tVE1/tVE1 skin transcriptome,SAMEA3880685,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,ENA first public:2016 05 24|ENA last update:2016 02 25|External Id:SAMEA3880685|INSDC center alias:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC center name:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC first public:2016 05 24T17:02:01Z|INSDC last update:2016 02 25T18:42:06Z|INSDC status:public|Submitter Id:VE1|allele:mau tVE1/tVE1|common name:zebrafish|dev stage:adult|sample name:VE1|tissue type:skin,,,,,,,,,Illumina HiSeq 2000 sequencing,ena EXPERIMENT MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 25 02 2016 18:41:58:669 5,ve12,1,Illumina TruSeq RNA,,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2000,,ERP014370,Illumina HiSeq 2000 sequencing,ENA FIRST PUBLIC:2016 05 24|ENA LAST UPDATE:2018 11 16,ve12_ATGTCA_L008_R1_001.fastq.gz,fastq,1446322222.0,14320022.0,ena RUN MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 25 02 2016 18:41:58:669 5,0:101,A:400821160;C:324210104;G:320286425;T:400078485;N:926048,101,,,,400821160,324210104,320286425,400078485,926048,ERX1365621,ERS1067819,ERA567249,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|European Nucleotide Archive,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,1,0.92778,,0.09489,,0.73093,,0.48493,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2016-02-25,Adult,Adult,Skin,Surface Structure
3764,ERR1294274,ERX1365620,ERS1067819,ERP014370,PRJEB12848,Danio rerio Mau mutants skin Transcriptome,ena-STUDY-MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY-25-02-2016-18:42:00:594-265,Other,Mau/Aqp3a dominant mutants of zebrafish Danio rerio are characterized by broken stripes and short fins. To identify signaling pathways affected by mau mutations and leading to pigment patterning defects we analyze skin transcriptome of adult wild type TU fish and two mau alleles.,ENA FIRST PUBLIC:2016 05 24|ENA LAST UPDATE:2016 02 25,,,Mau tVE1/tVE1 skin transcriptome,SAMEA3880685,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,ENA first public:2016 05 24|ENA last update:2016 02 25|External Id:SAMEA3880685|INSDC center alias:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC center name:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC first public:2016 05 24T17:02:01Z|INSDC last update:2016 02 25T18:42:06Z|INSDC status:public|Submitter Id:VE1|allele:mau tVE1/tVE1|common name:zebrafish|dev stage:adult|sample name:VE1|tissue type:skin,,,,,,,,,Illumina HiSeq 2000 sequencing,ena EXPERIMENT MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 25 02 2016 18:41:58:669 4,ve11,1,Illumina TruSeq RNA,,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2000,,ERP014370,Illumina HiSeq 2000 sequencing,ENA FIRST PUBLIC:2016 05 24|ENA LAST UPDATE:2018 11 16,ve11_AGTTCC_L008_R1_001.fastq.gz,fastq,2117238861.0,20962761.0,ena RUN MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 25 02 2016 18:41:58:669 4,0:101,A:591654664;C:469803769;G:462550117;T:591863795;N:1366516,101,,,,591654664,469803769,462550117,591863795,1366516,ERX1365620,ERS1067819,ERA567249,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|European Nucleotide Archive,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,1,0.93146,,0.09666,,0.73501,,0.48749,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2016-02-25,Adult,Adult,Skin,Surface Structure
3765,ERR1294273,ERX1365619,ERS1067818,ERP014370,PRJEB12848,Danio rerio Mau mutants skin Transcriptome,ena-STUDY-MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY-25-02-2016-18:42:00:594-265,Other,Mau/Aqp3a dominant mutants of zebrafish Danio rerio are characterized by broken stripes and short fins. To identify signaling pathways affected by mau mutations and leading to pigment patterning defects we analyze skin transcriptome of adult wild type TU fish and two mau alleles.,ENA FIRST PUBLIC:2016 05 24|ENA LAST UPDATE:2016 02 25,,,WT skin transcriptome,SAMEA3880684,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,ENA first public:2016 05 24|ENA last update:2016 02 25|External Id:SAMEA3880684|INSDC center alias:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC center name:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC first public:2016 05 24T17:02:01Z|INSDC last update:2016 02 25T18:42:06Z|INSDC status:public|Submitter Id:WT|allele:mau +/+|common name:zebrafish|dev stage:adult|sample name:WT|tissue type:skin,,,,,,,,,Illumina HiSeq 2000 sequencing,ena EXPERIMENT MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 25 02 2016 18:41:58:669 3,wt3,1,Illumina TruSeq RNA,,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2000,,ERP014370,Illumina HiSeq 2000 sequencing,ENA FIRST PUBLIC:2016 05 24|ENA LAST UPDATE:2018 11 16,wt3_AGTCAA_L008_R1_001.fastq.gz,fastq,2631687411.0,26056311.0,ena RUN MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 25 02 2016 18:41:58:669 3,0:101,A:734843629;C:584316000;G:577487406;T:733356850;N:1683526,101,,,,734843629,584316000,577487406,733356850,1683526,ERX1365619,ERS1067818,ERA567249,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|European Nucleotide Archive,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,1,0.95114,,0.1036,,0.72474,,0.48261,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2016-02-25,Adult,Adult,Skin,Surface Structure
3766,ERR1294272,ERX1365618,ERS1067818,ERP014370,PRJEB12848,Danio rerio Mau mutants skin Transcriptome,ena-STUDY-MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY-25-02-2016-18:42:00:594-265,Other,Mau/Aqp3a dominant mutants of zebrafish Danio rerio are characterized by broken stripes and short fins. To identify signaling pathways affected by mau mutations and leading to pigment patterning defects we analyze skin transcriptome of adult wild type TU fish and two mau alleles.,ENA FIRST PUBLIC:2016 05 24|ENA LAST UPDATE:2016 02 25,,,WT skin transcriptome,SAMEA3880684,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,ENA first public:2016 05 24|ENA last update:2016 02 25|External Id:SAMEA3880684|INSDC center alias:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC center name:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC first public:2016 05 24T17:02:01Z|INSDC last update:2016 02 25T18:42:06Z|INSDC status:public|Submitter Id:WT|allele:mau +/+|common name:zebrafish|dev stage:adult|sample name:WT|tissue type:skin,,,,,,,,,Illumina HiSeq 2000 sequencing,ena EXPERIMENT MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 25 02 2016 18:41:58:669 2,wt2,1,Illumina TruSeq RNA,,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2000,,ERP014370,Illumina HiSeq 2000 sequencing,ENA FIRST PUBLIC:2016 05 24|ENA LAST UPDATE:2018 11 16,wt2_CTTGTA_L008_R1_001.fastq.gz,fastq,2093443463.0,20727163.0,ena RUN MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 25 02 2016 18:41:58:669 2,0:101,A:607582034;C:442802229;G:437957089;T:603752294;N:1349817,101,,,,607582034,442802229,437957089,603752294,1349817,ERX1365618,ERS1067818,ERA567249,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|European Nucleotide Archive,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,1,0.94792,,0.10892,,0.73277,,0.51067,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2016-02-25,Adult,Adult,Skin,Surface Structure
3767,ERR1294271,ERX1365617,ERS1067818,ERP014370,PRJEB12848,Danio rerio Mau mutants skin Transcriptome,ena-STUDY-MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY-25-02-2016-18:42:00:594-265,Other,Mau/Aqp3a dominant mutants of zebrafish Danio rerio are characterized by broken stripes and short fins. To identify signaling pathways affected by mau mutations and leading to pigment patterning defects we analyze skin transcriptome of adult wild type TU fish and two mau alleles.,ENA FIRST PUBLIC:2016 05 24|ENA LAST UPDATE:2016 02 25,,,WT skin transcriptome,SAMEA3880684,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,ENA first public:2016 05 24|ENA last update:2016 02 25|External Id:SAMEA3880684|INSDC center alias:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC center name:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC first public:2016 05 24T17:02:01Z|INSDC last update:2016 02 25T18:42:06Z|INSDC status:public|Submitter Id:WT|allele:mau +/+|common name:zebrafish|dev stage:adult|sample name:WT|tissue type:skin,,,,,,,,,Illumina HiSeq 2000 sequencing,ena EXPERIMENT MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 25 02 2016 18:41:58:668 1,wt1,1,Illumina TruSeq RNA,,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,SINGLE,ILLUMINA,Illumina HiSeq 2000,,ERP014370,Illumina HiSeq 2000 sequencing,ENA FIRST PUBLIC:2016 05 24|ENA LAST UPDATE:2018 11 16,wt1_CAGATC_L008_R1_001.fastq.gz,fastq,1701561241.0,16847141.0,ena RUN MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 25 02 2016 18:41:58:668 1,0:101,A:505358711;C:348893979;G:343445186;T:502880170;N:983195,101,,,,505358711,348893979,343445186,502880170,983195,ERX1365617,ERS1067818,ERA567249,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|European Nucleotide Archive,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,1,0.94565,,0.13032,,0.73669,,0.50473,,101,,B,,usable mapping rate,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2016-02-25,Adult,Adult,Skin,Surface Structure
9770,ERR5838122,ERX5487778,ERS6337138,ERP119543,PRJEB36360,Transcriptomic analysis of adult skin from 9 Danio species,ena-STUDY-MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY-22-01-2020-08:38:04:562-27,Other,Transcriptomic analysis was carried out on adult skin of the flanks for the following nine Danio species: Danio rerio Danio aesculapii Danio nigrofasciatus Danio tinwini Danio kyathit Danio albolineatus Danio choprae Danio margaritatus and Danio erythromicron. Five replicates per species were carried out. Each replicate was derived from a single fish with skin samples coming from both flanks. Paired end stranded RNA Seq was then carried out.,ENA FIRST PUBLIC:2020 03 21|ENA LAST UPDATE:2021 04 21,PUBMED:33277491,Transcriptomic analysis was carried out on adult skin of the flanks for the following nine Danio species: Danio rerio Danio aesculapii Danio nigrofasciatus Danio tinwini Danio kyathit Danio albolineatus Danio choprae Danio margaritatus and Danio erythromicron. Five replicates per species were carried out. Each replicate was derived from a single fish with skin samples coming from both flanks. Paired end stranded RNA Seq was then carried out.,Transcriptomic analysis of adult skin from 9 Danio species,SAMEA8652584,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,ENA first public:2021 04 30|ENA last update:2021 04 29|External Id:SAMEA8652584|INSDC center alias:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC center name:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC first public:2021 04 30T00:30:20Z|INSDC last update:2021 04 29T02:45:00Z|INSDC status:public|Submitter Id:05 Danio rerio adult skin 5|collected by:Marco Podobnik|collection date:2019 08 23|common name:zebrafish|dev stage:adult|identified by:Marco Podobnik|isolation source:Tuebingen|sample name:05 Danio rerio adult skin 5|sex:male|tissue type:skin,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing,ena EXPERIMENT MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 28 04 2021 08:27:34:651 5,unspecified,1,TruSeq stranded mRNA Illumina,,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP119543,Illumina NovaSeq 6000 paired end sequencing,ENA FIRST PUBLIC:2021 04 30|ENA LAST UPDATE:2021 04 29,S1385Nr5.1.fastq.gz S1385Nr5.2.fastq.gz,fastq fastq,6912206316.0,34306523.0,ena RUN MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 28 04 2021 08:27:34:651 5,0:100.74 1:100.74,A:1853779551;C:1607929988;G:1658402611;T:1792045990;N:48176,100,100,,,1853779551,1607929988,1658402611,1792045990,48176,ERX5487778,ERS6337138,ERA4129786,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|European Nucleotide Archive,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,2,0.96095,0.962,0.08049,0.07821,0.70654,0.70926,0.51398,0.51064,101,101,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2020-03-21,Adult,Adult,Skin,Surface Structure
9771,ERR5838121,ERX5487777,ERS6337136,ERP119543,PRJEB36360,Transcriptomic analysis of adult skin from 9 Danio species,ena-STUDY-MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY-22-01-2020-08:38:04:562-27,Other,Transcriptomic analysis was carried out on adult skin of the flanks for the following nine Danio species: Danio rerio Danio aesculapii Danio nigrofasciatus Danio tinwini Danio kyathit Danio albolineatus Danio choprae Danio margaritatus and Danio erythromicron. Five replicates per species were carried out. Each replicate was derived from a single fish with skin samples coming from both flanks. Paired end stranded RNA Seq was then carried out.,ENA FIRST PUBLIC:2020 03 21|ENA LAST UPDATE:2021 04 21,PUBMED:33277491,Transcriptomic analysis was carried out on adult skin of the flanks for the following nine Danio species: Danio rerio Danio aesculapii Danio nigrofasciatus Danio tinwini Danio kyathit Danio albolineatus Danio choprae Danio margaritatus and Danio erythromicron. Five replicates per species were carried out. Each replicate was derived from a single fish with skin samples coming from both flanks. Paired end stranded RNA Seq was then carried out.,Transcriptomic analysis of adult skin from 9 Danio species,SAMEA8652582,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,ENA first public:2021 04 30|ENA last update:2021 04 29|External Id:SAMEA8652582|INSDC center alias:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC center name:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC first public:2021 04 30T00:30:20Z|INSDC last update:2021 04 29T02:45:00Z|INSDC status:public|Submitter Id:04 Danio rerio adult skin 4|collected by:Marco Podobnik|collection date:2019 08 23|common name:zebrafish|dev stage:adult|identified by:Marco Podobnik|isolation source:Tuebingen|sample name:04 Danio rerio adult skin 4|sex:male|tissue type:skin,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing,ena EXPERIMENT MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 28 04 2021 08:27:34:651 4,unspecified,1,TruSeq stranded mRNA Illumina,,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP119543,Illumina NovaSeq 6000 paired end sequencing,ENA FIRST PUBLIC:2021 04 30|ENA LAST UPDATE:2021 04 29,S1385Nr4.1.fastq.gz S1385Nr4.2.fastq.gz,fastq fastq,6161942050.0,30595009.0,ena RUN MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 28 04 2021 08:27:34:651 4,0:100.70 1:100.70,A:1642716052;C:1446485458;G:1490121106;T:1582576605;N:42829,100,100,,,1642716052,1446485458,1490121106,1582576605,42829,ERX5487777,ERS6337136,ERA4129786,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|European Nucleotide Archive,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,2,0.96544,0.96564,0.07368,0.07303,0.70822,0.71092,0.50154,0.50781,101,101,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2020-03-21,Adult,Adult,Skin,Surface Structure
9772,ERR5838120,ERX5487776,ERS6337133,ERP119543,PRJEB36360,Transcriptomic analysis of adult skin from 9 Danio species,ena-STUDY-MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY-22-01-2020-08:38:04:562-27,Other,Transcriptomic analysis was carried out on adult skin of the flanks for the following nine Danio species: Danio rerio Danio aesculapii Danio nigrofasciatus Danio tinwini Danio kyathit Danio albolineatus Danio choprae Danio margaritatus and Danio erythromicron. Five replicates per species were carried out. Each replicate was derived from a single fish with skin samples coming from both flanks. Paired end stranded RNA Seq was then carried out.,ENA FIRST PUBLIC:2020 03 21|ENA LAST UPDATE:2021 04 21,PUBMED:33277491,Transcriptomic analysis was carried out on adult skin of the flanks for the following nine Danio species: Danio rerio Danio aesculapii Danio nigrofasciatus Danio tinwini Danio kyathit Danio albolineatus Danio choprae Danio margaritatus and Danio erythromicron. Five replicates per species were carried out. Each replicate was derived from a single fish with skin samples coming from both flanks. Paired end stranded RNA Seq was then carried out.,Transcriptomic analysis of adult skin from 9 Danio species,SAMEA8652579,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,ENA first public:2021 04 30|ENA last update:2021 04 29|External Id:SAMEA8652579|INSDC center alias:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC center name:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC first public:2021 04 30T00:30:20Z|INSDC last update:2021 04 29T02:45:00Z|INSDC status:public|Submitter Id:03 Danio rerio adult skin 3|collected by:Marco Podobnik|collection date:2019 08 23|common name:zebrafish|dev stage:adult|identified by:Marco Podobnik|isolation source:Tuebingen|sample name:03 Danio rerio adult skin 3|sex:female|tissue type:skin,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing,ena EXPERIMENT MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 28 04 2021 08:27:34:651 3,unspecified,1,TruSeq stranded mRNA Illumina,,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP119543,Illumina NovaSeq 6000 paired end sequencing,ENA FIRST PUBLIC:2021 04 30|ENA LAST UPDATE:2021 04 29,S1385Nr3.1.fastq.gz S1385Nr3.2.fastq.gz,fastq fastq,7422881394.0,36866805.0,ena RUN MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 28 04 2021 08:27:34:651 3,0:100.67 1:100.67,A:1950275527;C:1768648993;G:1827362519;T:1876542858;N:51497,100,100,,,1950275527,1768648993,1827362519,1876542858,51497,ERX5487776,ERS6337133,ERA4129786,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|European Nucleotide Archive,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,2,0.97031,0.97047,0.04915,0.04817,0.71459,0.71894,0.51089,0.5097,101,101,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2020-03-21,Adult,Adult,Skin,Surface Structure
9773,ERR5838119,ERX5487775,ERS6337132,ERP119543,PRJEB36360,Transcriptomic analysis of adult skin from 9 Danio species,ena-STUDY-MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY-22-01-2020-08:38:04:562-27,Other,Transcriptomic analysis was carried out on adult skin of the flanks for the following nine Danio species: Danio rerio Danio aesculapii Danio nigrofasciatus Danio tinwini Danio kyathit Danio albolineatus Danio choprae Danio margaritatus and Danio erythromicron. Five replicates per species were carried out. Each replicate was derived from a single fish with skin samples coming from both flanks. Paired end stranded RNA Seq was then carried out.,ENA FIRST PUBLIC:2020 03 21|ENA LAST UPDATE:2021 04 21,PUBMED:33277491,Transcriptomic analysis was carried out on adult skin of the flanks for the following nine Danio species: Danio rerio Danio aesculapii Danio nigrofasciatus Danio tinwini Danio kyathit Danio albolineatus Danio choprae Danio margaritatus and Danio erythromicron. Five replicates per species were carried out. Each replicate was derived from a single fish with skin samples coming from both flanks. Paired end stranded RNA Seq was then carried out.,Transcriptomic analysis of adult skin from 9 Danio species,SAMEA8652578,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,ENA first public:2021 04 30|ENA last update:2021 04 29|External Id:SAMEA8652578|INSDC center alias:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC center name:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC first public:2021 04 30T00:30:20Z|INSDC last update:2021 04 29T02:45:00Z|INSDC status:public|Submitter Id:02 Danio rerio adult skin 2|collected by:Marco Podobnik|collection date:2019 08 23|common name:zebrafish|dev stage:adult|identified by:Marco Podobnik|isolation source:Tuebingen|sample name:02 Danio rerio adult skin 2|sex:female|tissue type:skin,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing,ena EXPERIMENT MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 28 04 2021 08:27:34:651 2,unspecified,1,TruSeq stranded mRNA Illumina,,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP119543,Illumina NovaSeq 6000 paired end sequencing,ENA FIRST PUBLIC:2021 04 30|ENA LAST UPDATE:2021 04 29,S1385Nr2.1.fastq.gz S1385Nr2.2.fastq.gz,fastq fastq,7006718028.0,34786110.0,ena RUN MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 28 04 2021 08:27:34:651 2,0:100.71 1:100.71,A:1847371523;C:1665346769;G:1712373266;T:1781565768;N:60702,100,100,,,1847371523,1665346769,1712373266,1781565768,60702,ERX5487775,ERS6337132,ERA4129786,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|European Nucleotide Archive,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,2,0.96136,0.96039,0.05872,0.05752,0.70325,0.70674,0.46717,0.48218,101,101,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2020-03-21,Adult,Adult,Skin,Surface Structure
9774,ERR5838118,ERX5487774,ERS6337063,ERP119543,PRJEB36360,Transcriptomic analysis of adult skin from 9 Danio species,ena-STUDY-MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY-22-01-2020-08:38:04:562-27,Other,Transcriptomic analysis was carried out on adult skin of the flanks for the following nine Danio species: Danio rerio Danio aesculapii Danio nigrofasciatus Danio tinwini Danio kyathit Danio albolineatus Danio choprae Danio margaritatus and Danio erythromicron. Five replicates per species were carried out. Each replicate was derived from a single fish with skin samples coming from both flanks. Paired end stranded RNA Seq was then carried out.,ENA FIRST PUBLIC:2020 03 21|ENA LAST UPDATE:2021 04 21,PUBMED:33277491,Transcriptomic analysis was carried out on adult skin of the flanks for the following nine Danio species: Danio rerio Danio aesculapii Danio nigrofasciatus Danio tinwini Danio kyathit Danio albolineatus Danio choprae Danio margaritatus and Danio erythromicron. Five replicates per species were carried out. Each replicate was derived from a single fish with skin samples coming from both flanks. Paired end stranded RNA Seq was then carried out.,Transcriptomic analysis of adult skin from 9 Danio species,SAMEA8652509,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,ENA first public:2021 04 30|ENA last update:2021 04 29|External Id:SAMEA8652509|INSDC center alias:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC center name:MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|INSDC first public:2021 04 30T00:30:20Z|INSDC last update:2021 04 29T02:45:00Z|INSDC status:public|Submitter Id:01 Danio rerio adult skin 1|collected by:Marco Podobnik|collection date:2019 08 23|common name:zebrafish|dev stage:adult|identified by:Marco Podobnik|isolation source:Tuebingen|sample name:01 Danio rerio adult skin 1|sex:male|tissue type:skin,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing,ena EXPERIMENT MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 28 04 2021 08:27:34:651 1,unspecified,1,TruSeq stranded mRNA Illumina,,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP119543,Illumina NovaSeq 6000 paired end sequencing,ENA FIRST PUBLIC:2021 04 30|ENA LAST UPDATE:2021 04 29,S1385Nr1.1.fastq.gz S1385Nr1.2.fastq.gz,fastq fastq,6586457266.0,32719900.0,ena RUN MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY 28 04 2021 08:27:34:651 1,0:100.65 1:100.65,A:1709547533;C:1593125315;G:1617088678;T:1666650824;N:44916,100,100,,,1709547533,1593125315,1617088678,1666650824,44916,ERX5487774,ERS6337063,ERA4129786,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY|European Nucleotide Archive,MAX PLANCK INSTITUTE FOR DEVELOPMENTAL BIOLOGY,2,0.9531,0.95366,0.07763,0.07567,0.71384,0.71634,0.52409,0.52475,101,101,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,Germany,2020-03-21,Adult,Adult,Skin,Surface Structure
33837,SRR30763634,SRX26165999,SRS22710780,SRP534093,PRJNA1163532,Skin as outermost immune organ of vertebrates that elicits robust early immune responses post immunization with glycoprotein of spring viraemia of carp virus,PRJNA1163532,Whole Genome Sequencing,,,,,Skin con2,Skin con 3,,breed:zebrafish|age:6 month|collection date:2020 01 10|geo loc name:China: Yangling|sex:not collected|tissue:skin|treatment:Skin con 3|BioSampleModel:Model organism or animal,,,,,,,,,Zebrafish skin RNA seq Skin con 3,SK CON 3,SK CON 3,Zebrafish skin at control group,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP534093,,,Skin_con2_Clean_Data1.fq.gz Skin_con2_Clean_Data2.fq.gz,fastq fastq,6083362246.0,21717347.0,Skin con2 Clean Data1.fq.gz,0:140.06 1:140.05,A:1575684711;C:1458969719;G:1469933956;T:1578641539;N:132321,140,140,,,1575684711,1458969719,1469933956,1578641539,132321,SRX26165999,SRS22710780,SRA1977051,Northwest A&F University|College of Animal Science and Technology,Northwest A&F University,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,unknown,unknown,,China,2024-09-22,Adult,Adult,Skin,Surface Structure
33838,SRR30763635,SRX26165998,SRS22710779,SRP534093,PRJNA1163532,Skin as outermost immune organ of vertebrates that elicits robust early immune responses post immunization with glycoprotein of spring viraemia of carp virus,PRJNA1163532,Whole Genome Sequencing,,,,,Skin con1,Skin con 2,,breed:zebrafish|age:6 month|collection date:2020 01 10|geo loc name:China: Yangling|sex:not collected|tissue:skin|treatment:Skin con 2|BioSampleModel:Model organism or animal,,,,,,,,,Zebrafish skin RNA seq Skin con 2,SK CON 2,SK CON 2,Zebrafish skin at control group,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP534093,,,Skin_con1_Clean_Data1.fq.gz Skin_con1_Clean_Data2.fq.gz,fastq fastq,5776524166.0,20610041.0,Skin con1 Clean Data1.fq.gz,0:140.14 1:140.13,A:1496887200;C:1385008310;G:1395131090;T:1499373290;N:124276,140,140,,,1496887200,1385008310,1395131090,1499373290,124276,SRX26165998,SRS22710779,SRA1977051,Northwest A&F University|College of Animal Science and Technology,Northwest A&F University,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,unknown,unknown,,China,2024-09-22,Adult,Adult,Skin,Surface Structure
33839,SRR30763636,SRX26165997,SRS22710778,SRP534093,PRJNA1163532,Skin as outermost immune organ of vertebrates that elicits robust early immune responses post immunization with glycoprotein of spring viraemia of carp virus,PRJNA1163532,Whole Genome Sequencing,,,,,Skin con,Skin con 1,,breed:zebrafish|age:6 month|collection date:2020 01 10|geo loc name:China: Yangling|sex:not collected|tissue:skin|treatment:Skin con 1|BioSampleModel:Model organism or animal,,,,,,,,,Zebrafish skin RNA seq Skin con 1,SK CON 1,SK CON 1,Zebrafish skin at control group,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP534093,,,Skin_con_Clean_Data1.fq.gz Skin_con_Clean_Data2.fq.gz,fastq fastq,5304184337.0,18925504.0,Skin con Clean Data1.fq.gz,0:140.14 1:140.13,A:1400015654;C:1245459230;G:1267684230;T:1391013551;N:11672,140,140,,,1400015654,1245459230,1267684230,1391013551,11672,SRX26165997,SRS22710778,SRA1977051,Northwest A&F University|College of Animal Science and Technology,Northwest A&F University,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,unknown,unknown,,China,2024-09-22,Adult,Adult,Skin,Surface Structure
33840,SRR30763637,SRX26165996,SRS22710777,SRP534093,PRJNA1163532,Skin as outermost immune organ of vertebrates that elicits robust early immune responses post immunization with glycoprotein of spring viraemia of carp virus,PRJNA1163532,Whole Genome Sequencing,,,,,Skin 3dpi2,Skin 3d 3,,breed:zebrafish|age:6 month|collection date:2020 01 10|geo loc name:China: Yangling|sex:not collected|tissue:skin|treatment:Skin 3dpi 3|BioSampleModel:Model organism or animal,,,,,,,,,Zebrafish skin RNA seq Skin 3d 3,SK 3D 3,SK 3D 3,Zebrafish skin at 3 dpv post immunization with glycoprotein of spring viraemia of carp virus,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP534093,,,Skin_3dpi2_Clean_Data1.fq.gz Skin_3dpi2_Clean_Data2.fq.gz,fastq fastq,5528962547.0,19717071.0,Skin 3dpi2 Clean Data1.fq.gz,0:140.21 1:140.20,A:1434889776;C:1319598036;G:1335801562;T:1438550030;N:123143,140,140,,,1434889776,1319598036,1335801562,1438550030,123143,SRX26165996,SRS22710777,SRA1977051,Northwest A&F University|College of Animal Science and Technology,Northwest A&F University,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,unknown,unknown,,China,2024-09-22,Adult,Adult,Skin,Surface Structure
33841,SRR30763638,SRX26165995,SRS22710776,SRP534093,PRJNA1163532,Skin as outermost immune organ of vertebrates that elicits robust early immune responses post immunization with glycoprotein of spring viraemia of carp virus,PRJNA1163532,Whole Genome Sequencing,,,,,Skin 3dpi1,Skin 3d 2,,breed:zebrafish|age:6 month|collection date:2020 01 10|geo loc name:China: Yangling|sex:not collected|tissue:skin|treatment:Skin 3dpi 2|BioSampleModel:Model organism or animal,,,,,,,,,Zebrafish skin RNA seq Skin 3d 2,SK 3D 2,SK 3D 2,Zebrafish skin at 3 dpv post immunization with glycoprotein of spring viraemia of carp virus,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP534093,,,Skin_3dpi1_Clean_Data1.fq.gz Skin_3dpi1_Clean_Data2.fq.gz,fastq fastq,6045424568.0,21559158.0,Skin 3dpi1 Clean Data1.fq.gz,0:140.21 1:140.20,A:1566211023;C:1442845401;G:1464489674;T:1571745680;N:132790,140,140,,,1566211023,1442845401,1464489674,1571745680,132790,SRX26165995,SRS22710776,SRA1977051,Northwest A&F University|College of Animal Science and Technology,Northwest A&F University,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,unknown,unknown,,China,2024-09-22,Adult,Adult,Skin,Surface Structure
33842,SRR30763639,SRX26165994,SRS22710775,SRP534093,PRJNA1163532,Skin as outermost immune organ of vertebrates that elicits robust early immune responses post immunization with glycoprotein of spring viraemia of carp virus,PRJNA1163532,Whole Genome Sequencing,,,,,Skin 3dpi,Skin 3d 1,,breed:zebrafish|age:6 month|collection date:2020 01 10|geo loc name:China: Yangling|sex:not collected|tissue:skin|treatment:Skin 3dpi 1|BioSampleModel:Model organism or animal,,,,,,,,,Zebrafish skin RNA seq Skin 3d 1,SK 3D 1,SK 3D 1,Zebrafish skin at 3 dpv post immunization with glycoprotein of spring viraemia of carp virus,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP534093,,,Skin_3dpi_Clean_Data1.fq.gz Skin_3dpi_Clean_Data2.fq.gz,fastq fastq,5398897838.0,19388025.0,Skin 3dpi Clean Data1.fq.gz,0:139.24 1:139.23,A:1434301676;C:1260747084;G:1277759027;T:1426077485;N:12566,139,139,,,1434301676,1260747084,1277759027,1426077485,12566,SRX26165994,SRS22710775,SRA1977051,Northwest A&F University|College of Animal Science and Technology,Northwest A&F University,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,unknown,unknown,,China,2024-09-22,Adult,Adult,Skin,Surface Structure
33843,SRR30763640,SRX26165993,SRS22710774,SRP534093,PRJNA1163532,Skin as outermost immune organ of vertebrates that elicits robust early immune responses post immunization with glycoprotein of spring viraemia of carp virus,PRJNA1163532,Whole Genome Sequencing,,,,,Skin 1dpi2,Skin 1d 3,,breed:zebrafish|age:6 month|collection date:2020 01 10|geo loc name:China: Yangling|sex:not collected|tissue:skin|treatment:Skin 1dpi 3|BioSampleModel:Model organism or animal,,,,,,,,,Zebrafish skin RNA seq Skin 1d 3,SK 1D 3,SK 1D 3,Zebrafish skin at 1 dpv post immunization with glycoprotein of spring viraemia of carp virus,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP534093,,,Skin_1dpi2_Clean_Data1.fq.gz Skin_1dpi2_Clean_Data2.fq.gz,fastq fastq,6317753535.0,22527726.0,Skin 1dpi2 Clean Data1.fq.gz,0:140.23 1:140.22,A:1637469629;C:1508203339;G:1526532646;T:1645411954;N:135967,140,140,,,1637469629,1508203339,1526532646,1645411954,135967,SRX26165993,SRS22710774,SRA1977051,Northwest A&F University|College of Animal Science and Technology,Northwest A&F University,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,unknown,unknown,,China,2024-09-22,Adult,Adult,Skin,Surface Structure
33844,SRR30763641,SRX26165992,SRS22710773,SRP534093,PRJNA1163532,Skin as outermost immune organ of vertebrates that elicits robust early immune responses post immunization with glycoprotein of spring viraemia of carp virus,PRJNA1163532,Whole Genome Sequencing,,,,,Skin 1dpi1,Skin 1d 2,,breed:zebrafish|age:6 month|collection date:2020 01 10|geo loc name:China: Yangling|sex:not collected|tissue:skin|treatment:Skin 1dpi 2|BioSampleModel:Model organism or animal,,,,,,,,,Zebrafish skin RNA seq Skin 1d 2,SK 1D 2,SK 1D 2,Zebrafish skin at 1 dpv post immunization with glycoprotein of spring viraemia of carp virus,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP534093,,,Skin_1dpi1_Clean_Data1.fq.gz Skin_1dpi1_Clean_Data2.fq.gz,fastq fastq,5665401221.0,20209478.0,Skin 1dpi1 Clean Data1.fq.gz,0:140.17 1:140.16,A:1463485728;C:1359900876;G:1372085350;T:1469804976;N:124291,140,140,,,1463485728,1359900876,1372085350,1469804976,124291,SRX26165992,SRS22710773,SRA1977051,Northwest A&F University|College of Animal Science and Technology,Northwest A&F University,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,unknown,unknown,,China,2024-09-22,Adult,Adult,Skin,Surface Structure
33845,SRR30763642,SRX26165991,SRS22710772,SRP534093,PRJNA1163532,Skin as outermost immune organ of vertebrates that elicits robust early immune responses post immunization with glycoprotein of spring viraemia of carp virus,PRJNA1163532,Whole Genome Sequencing,,,,,Skin 1dpi,Skin 1d 1,,breed:zebrafish|age:6 month|collection date:2020 01 10|geo loc name:China: Yangling|sex:not collected|tissue:skin|treatment:Skin 1dpi 1|BioSampleModel:Model organism or animal,,,,,,,,,Zebrafish skin RNA seq Skin 1d 1,SK 1D 1,SK 1D 1,Zebrafish skin at 1 dpv post immunization with glycoprotein of spring viraemia of carp virus,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP534093,,,Skin_1dpi_Clean_Data1.fq.gz Skin_1dpi_Clean_Data2.fq.gz,fastq fastq,4758945020.0,17104843.0,Skin 1dpi Clean Data1.fq.gz,0:139.12 1:139.11,A:1261511064;C:1113177324;G:1128285977;T:1255959998;N:10657,139,139,,,1261511064,1113177324,1128285977,1255959998,10657,SRX26165991,SRS22710772,SRA1977051,Northwest A&F University|College of Animal Science and Technology,Northwest A&F University,,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,unknown,bulk,unknown,unknown,,China,2024-09-22,Adult,Adult,Skin,Surface Structure
36385,SRR516135,SRX156099,SRS346038,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB 5m skin mRNA,zebrafish Nr31 5m,,,,,,,,,,,1 male Danio rerio strain TueAB 5m skin mRNA Illumina GA IIx 76 bp,zebrafish Nr31 5m 1,zebrafish Nr31 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,760Application ReadForward1,SRP013931,,,,,1482677008.0,19508908.0,zebrafish Nr31 5m 1 1,0:76,A:385413939;C:354317020;G:354043481;T:388361606;N:540962,76,,,,385413939,354317020,354043481,388361606,540962,SRX156099,SRS346038,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.9273,,0.08335,,0.67898,,0.48956,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36386,SRR516140,SRX156099,SRS346038,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB 5m skin mRNA,zebrafish Nr31 5m,,,,,,,,,,,1 male Danio rerio strain TueAB 5m skin mRNA Illumina GA IIx 76 bp,zebrafish Nr31 5m 1,zebrafish Nr31 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,760Application ReadForward1,SRP013931,,,,,388560032.0,5112632.0,zebrafish Nr31 5m 1 2,0:76,A:102207965;C:91473514;G:92119606;T:102551859;N:207088,76,,,,102207965,91473514,92119606,102551859,207088,SRX156099,SRS346038,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92486,,0.08531,,0.67663,,0.49425,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36387,SRR516134,SRX156098,SRS346037,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB 5m skin mRNA,zebrafish Nr30 5m,,,,,,,,,,,1 male Danio rerio strain TueAB 5m skin mRNA Illumina GA IIx 76 bp,zebrafish Nr30 5m 1,zebrafish Nr30 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,760Application ReadForward1,SRP013931,,,,,1675582184.0,22047134.0,zebrafish Nr30 5m 1 1,0:76,A:458929887;C:381230210;G:376734439;T:457916852;N:770796,76,,,,458929887,381230210,376734439,457916852,770796,SRX156098,SRS346037,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91832,,0.10303,,0.71455,,0.49444,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36388,SRR516139,SRX156098,SRS346037,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB 5m skin mRNA,zebrafish Nr30 5m,,,,,,,,,,,1 male Danio rerio strain TueAB 5m skin mRNA Illumina GA IIx 76 bp,zebrafish Nr30 5m 1,zebrafish Nr30 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,760Application ReadForward1,SRP013931,,,,,477065908.0,6277183.0,zebrafish Nr30 5m 1 2,0:76,A:131927852;C:107181234;G:106214626;T:131489254;N:252942,76,,,,131927852,107181234,106214626,131489254,252942,SRX156098,SRS346037,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91581,,0.10754,,0.71577,,0.49994,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36389,SRR516133,SRX156097,SRS346036,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB 5m skin mRNA,zebrafish Nr29 5m,,,,,,,,,,,1 male Danio rerio strain TueAB 5m skin mRNA Illumina GA IIx 76 bp,zebrafish Nr29 5m 1,zebrafish Nr29 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,760Application ReadForward1,SRP013931,,,,,1305047832.0,17171682.0,zebrafish Nr29 5m 1 1,0:76,A:362467093;C:292043132;G:289641844;T:360441501;N:454262,76,,,,362467093,292043132,289641844,360441501,454262,SRX156097,SRS346036,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.90448,,0.11905,,0.70246,,0.47656,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36390,SRR516138,SRX156097,SRS346036,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB 5m skin mRNA,zebrafish Nr29 5m,,,,,,,,,,,1 male Danio rerio strain TueAB 5m skin mRNA Illumina GA IIx 76 bp,zebrafish Nr29 5m 1,zebrafish Nr29 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,760Application ReadForward1,SRP013931,,,110307_DanioRerio_Nr29_5m_w1_s_1.fq,fastq,457287972.0,6016947.0,zebrafish Nr29 5m 1 2,0:76,A:128212070;C:101000378;G:100136000;T:127695086;N:244438,76,,,,128212070,101000378,100136000,127695086,244438,SRX156097,SRS346036,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.90327,,0.12466,,0.70195,,0.49568,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2013-03-11,Adult,Adult,Skin,Surface Structure
36391,SRR516132,SRX156096,SRS346035,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB 5m skin mRNA,zebrafish Nr28 5m,,,,,,,,,,,1 male Danio rerio strain TueAB 5m skin mRNA Illumina GA IIx 76 bp,zebrafish Nr28 5m 1,zebrafish Nr28 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,760Application ReadForward1,SRP013931,,,110104_DanioRerio_Nr28_5m_l5.fq,fastq,778479248.0,10243148.0,zebrafish Nr28 5m 1 1,0:76,A:207103962;C:183338330;G:183353815;T:204420105;N:263036,76,,,,207103962,183338330,183353815,204420105,263036,SRX156096,SRS346035,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91711,,0.08403,,0.72218,,0.46568,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2013-03-11,Adult,Adult,Skin,Surface Structure
36392,SRR516137,SRX156096,SRS346035,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB 5m skin mRNA,zebrafish Nr28 5m,,,,,,,,,,,1 male Danio rerio strain TueAB 5m skin mRNA Illumina GA IIx 76 bp,zebrafish Nr28 5m 1,zebrafish Nr28 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,760Application ReadForward1,SRP013931,,,110307_DanioRerio_Nr28_5m_w1_s_2.fq,fastq,1004119144.0,13212094.0,zebrafish Nr28 5m 1 2,0:76,A:269579079;C:233472158;G:233160786;T:267709479;N:197642,76,,,,269579079,233472158,233160786,267709479,197642,SRX156096,SRS346035,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92125,,0.09157,,0.72056,,0.4782,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2013-03-11,Adult,Adult,Skin,Surface Structure
36393,SRR516131,SRX156095,SRS346034,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB 5m skin mRNA,zebrafish Nr27 5m,,,,,,,,,,,1 male Danio rerio strain TueAB 5m skin mRNA Illumina GA IIx 76 bp,zebrafish Nr27 5m 1,zebrafish Nr27 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,760Application ReadForward1,SRP013931,,,,,1689987528.0,22236678.0,zebrafish Nr27 5m 1 1,0:76,A:462852366;C:385867681;G:380635625;T:460044585;N:587271,76,,,,462852366,385867681,380635625,460044585,587271,SRX156095,SRS346034,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91288,,0.1015,,0.71293,,0.488,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36394,SRR516136,SRX156095,SRS346034,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB 5m skin mRNA,zebrafish Nr27 5m,,,,,,,,,,,1 male Danio rerio strain TueAB 5m skin mRNA Illumina GA IIx 76 bp,zebrafish Nr27 5m 1,zebrafish Nr27 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,760Application ReadForward1,SRP013931,,,,,421535824.0,5546524.0,zebrafish Nr27 5m 1 2,0:76,A:116434305;C:94958671;G:93876467;T:116039545;N:226836,76,,,,116434305,94958671,93876467,116039545,226836,SRX156095,SRS346034,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.90776,,0.10459,,0.71399,,0.49081,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36395,SRR516130,SRX156094,SRS346033,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB 5m skin mRNA,zebrafish Nr26 5m,,,,,,,,,,,1 male Danio rerio strain TueAB 5m skin mRNA Illumina GA IIx 76 bp,zebrafish Nr26 5m 1,zebrafish Nr26 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,760Application ReadForward1,SRP013931,,,,,1633478944.0,21493144.0,zebrafish Nr26 5m 1 1,0:76,A:435251153;C:384938751;G:380973255;T:432171855;N:143930,76,,,,435251153,384938751,380973255,432171855,143930,SRX156094,SRS346033,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92486,,0.08081,,0.71969,,0.50656,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36396,SRR516129,SRX156093,SRS346032,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB 5m skin mRNA,zebrafish Nr25 5m,,,,,,,,,,,1 male Danio rerio strain TueAB 5m skin mRNA Illumina GA IIx 76 bp,zebrafish Nr25 5m 1,zebrafish Nr25 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,760Application ReadForward1,SRP013931,,,101021_DanioRerio_Nr25_5m_l7.fq,fastq,1893537644.0,24914969.0,zebrafish Nr25 5m 1 1,0:76,A:516246577;C:431589332;G:431233057;T:514385629;N:83049,76,,,,516246577,431589332,431233057,514385629,83049,SRX156093,SRS346032,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.89513,,0.11188,,0.70591,,0.47827,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36397,SRR516128,SRX156092,SRS346031,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB 5m skin mRNA,zebrafish Nr23 5m,,,,,,,,,,,1 male Danio rerio strain TueAB 5m skin mRNA Illumina GA IIx 76 bp,zebrafish Nr23 5m 1,zebrafish Nr23 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,760Application ReadForward1,SRP013931,,,101021_DanioRerio_Nr23_5m_l6.fq,fastq,2271040360.0,29882110.0,zebrafish Nr23 5m 1 1,0:76,A:602084142;C:529733757;G:524301688;T:614827528;N:93245,76,,,,602084142,529733757,524301688,614827528,93245,SRX156092,SRS346031,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92194,,0.09655,,0.70463,,0.47059,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2013-03-11,Adult,Adult,Skin,Surface Structure
36398,SRR516127,SRX156091,SRS346030,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB 5m skin mRNA,zebrafish Nr22 5m,,,,,,,,,,,1 male Danio rerio strain TueAB 5m skin mRNA Illumina GA IIx 76 bp,zebrafish Nr22 5m 1,zebrafish Nr22 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,760Application ReadForward1,SRP013931,,,,,2812847324.0,37011149.0,zebrafish Nr22 5m 1 1,0:76,A:760320146;C:645617435;G:643672544;T:763112485;N:124714,76,,,,760320146,645617435,643672544,763112485,124714,SRX156091,SRS346030,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93272,,0.08997,,0.70755,,0.52272,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36399,SRR516126,SRX156090,SRS346029,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB 5m skin mRNA,zebrafish Nr21 5m,,,,,,,,,,,1 male Danio rerio strain TueAB 5m skin mRNA Illumina GA IIx 76 bp,zebrafish Nr21 5m 1,zebrafish Nr21 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,760Application ReadForward1,SRP013931,,,,,2331404728.0,30676378.0,zebrafish Nr21 5m 1 1,0:76,A:630276643;C:539444103;G:529126097;T:632442560;N:115325,76,,,,630276643,539444103,529126097,632442560,115325,SRX156090,SRS346029,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92747,,0.10341,,0.70414,,0.49626,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36400,SRR516125,SRX156089,SRS346028,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 3.5 years. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB 3.5y skin mRNA,zebrafish Nr18 3.5y,,,,,,,,,,,1 male Danio rerio strain TueAB 3.5y skin mRNA Illumina GA IIx 76 bp,zebrafish Nr18 3.5y 1,zebrafish Nr18 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,760Application ReadForward1,SRP013931,,,,,2653618888.0,34916038.0,zebrafish Nr18 3.5y 1 1,0:76,A:728660211;C:598216653;G:601771842;T:724871715;N:98467,76,,,,728660211,598216653,601771842,724871715,98467,SRX156089,SRS346028,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91227,,0.10895,,0.71802,,0.49946,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36401,SRR516124,SRX156088,SRS346027,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 3.5 years. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB 3.5y skin mRNA,zebrafish Nr14 3.5y,,,,,,,,,,,1 male Danio rerio strain TueAB 3.5y skin mRNA Illumina GA IIx 76 bp,zebrafish Nr14 3.5y 1,zebrafish Nr14 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,760Application ReadForward1,SRP013931,,,,,2013201012.0,26489487.0,zebrafish Nr14 3.5y 1 1,0:76,A:554778161;C:456082219;G:445029202;T:557232699;N:78731,76,,,,554778161,456082219,445029202,557232699,78731,SRX156088,SRS346027,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.90845,,0.1164,,0.71236,,0.48939,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36402,SRR516123,SRX156087,SRS346026,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 3.5 years. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB 3.5y skin mRNA,zebrafish Nr13 3.5y,,,,,,,,,,,1 male Danio rerio strain TueAB 3.5y skin mRNA Illumina GA IIx 76 bp,zebrafish Nr13 3.5y 1,zebrafish Nr13 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,760Application ReadForward1,SRP013931,,,,,2680273532.0,35266757.0,zebrafish Nr13 3.5y 1 1,0:76,A:740111520;C:588329849;G:606909161;T:744802954;N:120048,76,,,,740111520,588329849,606909161,744802954,120048,SRX156087,SRS346026,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.85899,,0.11949,,0.71595,,0.48655,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36403,SRR516122,SRX156086,SRS346025,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 3.5 years. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB 3.5y skin mRNA,zebrafish Nr12 3.5y,,,,,,,,,,,1 male Danio rerio strain TueAB 3.5y skin mRNA Illumina GA IIx 76 bp,zebrafish Nr12 3.5y 1,zebrafish Nr12 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,760Application ReadForward1,SRP013931,,,,,2982635200.0,39245200.0,zebrafish Nr12 3.5y 1 1,0:76,A:829678392;C:650275344;G:659157904;T:842323646;N:1199914,76,,,,829678392,650275344,659157904,842323646,1199914,SRX156086,SRS346025,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.88932,,0.12521,,0.71638,,0.53514,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36404,SRR516121,SRX156085,SRS346024,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 3.5 years. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB 3.5y skin mRNA,zebrafish Nr11 3.5y,,,,,,,,,,,1 male Danio rerio strain TueAB 3.5y skin mRNA Illumina GA IIx 76 bp,zebrafish Nr11 3.5y 1,zebrafish Nr11 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,760Application ReadForward1,SRP013931,,,,,2696915708.0,35485733.0,zebrafish Nr11 3.5y 1 1,0:76,A:749305835;C:599264137;G:607414252;T:740194221;N:737263,76,,,,749305835,599264137,607414252,740194221,737263,SRX156085,SRS346024,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.86902,,0.11557,,0.71461,,0.49255,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36712,SRR835167,SRX271963,SRS416259,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes Iridophores and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology we developed a method to concomitantly purify melanocytes iridophores and retinal pigmented epithelium from zebrafish and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium as well as in melanocytes and iridophores. We found 214 genes co enriched in melanocytes and retinal pigmented epithelium indicating the shared functions of melanin producing cells. We found 62 genes significantly co enriched in melanocytes and iridophores illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis phosphoribosyl pyrophosphate thus constituting a guanine cycle. The purification procedure and expression analysis described here along with the accompanying transcriptome wide expression data provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 116h 53,GSM1129625,,tissue:melanocytes|hpf,dm 116h 53,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013 based on NCBI's RefSeq build from 8/29/2012 current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129625,GSM1129625: dm 116h 53; Danio rerio; RNA Seq,GSM1129625 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129625,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_116h_53_440.fq.bz2,fastq,203842506.0,4853393.0,GSM1129625 r1,0:42,A:49784918;C:48871504;G:45835011;T:59338271;N:12802,42,,,,49784918,48871504,45835011,59338271,12802,SRX271963,SRS416259,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.82741,,0.14973,,0.84893,,0.53644,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36713,SRR835166,SRX271962,SRS416258,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes Iridophores and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology we developed a method to concomitantly purify melanocytes iridophores and retinal pigmented epithelium from zebrafish and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium as well as in melanocytes and iridophores. We found 214 genes co enriched in melanocytes and retinal pigmented epithelium indicating the shared functions of melanin producing cells. We found 62 genes significantly co enriched in melanocytes and iridophores illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis phosphoribosyl pyrophosphate thus constituting a guanine cycle. The purification procedure and expression analysis described here along with the accompanying transcriptome wide expression data provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 97h 41 440,GSM1129624,,tissue:melanocytes|hpf,dm 97h 41 440,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013 based on NCBI's RefSeq build from 8/29/2012 current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129624,GSM1129624: dm 97h 41 440; Danio rerio; RNA Seq,GSM1129624 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129624,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_97h_41_440.fq.bz2,fastq,199638768.0,4753304.0,GSM1129624 r1,0:42,A:45958194;C:50367228;G:48451580;T:54849238;N:12528,42,,,,45958194,50367228,48451580,54849238,12528,SRX271962,SRS416258,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.85741,,0.09897,,0.84112,,0.53299,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36714,SRR835165,SRX271961,SRS416257,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes Iridophores and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology we developed a method to concomitantly purify melanocytes iridophores and retinal pigmented epithelium from zebrafish and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium as well as in melanocytes and iridophores. We found 214 genes co enriched in melanocytes and retinal pigmented epithelium indicating the shared functions of melanin producing cells. We found 62 genes significantly co enriched in melanocytes and iridophores illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis phosphoribosyl pyrophosphate thus constituting a guanine cycle. The purification procedure and expression analysis described here along with the accompanying transcriptome wide expression data provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h FM 351,GSM1129623,,tissue:melanocytes|hpf,dm 77h FM 351,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013 based on NCBI's RefSeq build from 8/29/2012 current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129623,GSM1129623: dm 77h FM 351; Danio rerio; RNA Seq,GSM1129623 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129623,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_FM_351_ATT.fq.bz2,fastq,194385384.0,5399594.0,GSM1129623 r1,0:36,A:45805148;C:47931006;G:41165284;T:59482433;N:1513,36,,,,45805148,47931006,41165284,59482433,1513,SRX271961,SRS416257,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.77748,,0.07764,,0.87687,,0.51575,,36,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36715,SRR835164,SRX271960,SRS416256,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes Iridophores and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology we developed a method to concomitantly purify melanocytes iridophores and retinal pigmented epithelium from zebrafish and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium as well as in melanocytes and iridophores. We found 214 genes co enriched in melanocytes and retinal pigmented epithelium indicating the shared functions of melanin producing cells. We found 62 genes significantly co enriched in melanocytes and iridophores illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis phosphoribosyl pyrophosphate thus constituting a guanine cycle. The purification procedure and expression analysis described here along with the accompanying transcriptome wide expression data provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 52 440,GSM1129622,,tissue:melanocytes|hpf,dm 77h 52 440,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013 based on NCBI's RefSeq build from 8/29/2012 current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129622,GSM1129622: dm 77h 52 440; Danio rerio; RNA Seq,GSM1129622 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129622,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_52_440.fq.bz2,fastq,105047586.0,2501133.0,GSM1129622 r1,0:42,A:24711000;C:26459041;G:24869198;T:29001957;N:6390,42,,,,24711000,26459041,24869198,29001957,6390,SRX271960,SRS416256,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.88862,,0.09219,,0.83611,,0.55476,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36716,SRR835163,SRX271959,SRS416255,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes Iridophores and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology we developed a method to concomitantly purify melanocytes iridophores and retinal pigmented epithelium from zebrafish and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium as well as in melanocytes and iridophores. We found 214 genes co enriched in melanocytes and retinal pigmented epithelium indicating the shared functions of melanin producing cells. We found 62 genes significantly co enriched in melanocytes and iridophores illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis phosphoribosyl pyrophosphate thus constituting a guanine cycle. The purification procedure and expression analysis described here along with the accompanying transcriptome wide expression data provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 43 440,GSM1129621,,tissue:melanocytes|hpf,dm 77h 43 440,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013 based on NCBI's RefSeq build from 8/29/2012 current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129621,GSM1129621: dm 77h 43 440; Danio rerio; RNA Seq,GSM1129621 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129621,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_43_440.fq.bz2,fastq,371553084.0,8846502.0,GSM1129621 r1,0:42,A:84994407;C:91269417;G:87188701;T:108077735;N:22824,42,,,,84994407,91269417,87188701,108077735,22824,SRX271959,SRS416255,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.86495,,0.09748,,0.83684,,0.52833,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36717,SRR835162,SRX271958,SRS416254,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes Iridophores and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology we developed a method to concomitantly purify melanocytes iridophores and retinal pigmented epithelium from zebrafish and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium as well as in melanocytes and iridophores. We found 214 genes co enriched in melanocytes and retinal pigmented epithelium indicating the shared functions of melanin producing cells. We found 62 genes significantly co enriched in melanocytes and iridophores illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis phosphoribosyl pyrophosphate thus constituting a guanine cycle. The purification procedure and expression analysis described here along with the accompanying transcriptome wide expression data provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 28 399s62,GSM1129620,,tissue:melanocytes|hpf,dm 77h 28 399s62,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013 based on NCBI's RefSeq build from 8/29/2012 current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129620,GSM1129620: dm 77h 28 399s62; Danio rerio; RNA Seq,GSM1129620 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129620,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,,,731276158.0,3620179.0,GSM1129620 r1,0:101 1:101,A:173989639;C:199270484;G:188281318;T:169725601;N:9116,101,101,,,173989639,199270484,188281318,169725601,9116,SRX271958,SRS416254,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",2,0.84122,0.79964,0.02274,0.02168,0.89645,0.89073,0.63341,0.63015,101,101,B,B,biological fallback assumption,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36718,SRR835161,SRX271957,SRS416253,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes Iridophores and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology we developed a method to concomitantly purify melanocytes iridophores and retinal pigmented epithelium from zebrafish and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium as well as in melanocytes and iridophores. We found 214 genes co enriched in melanocytes and retinal pigmented epithelium indicating the shared functions of melanin producing cells. We found 62 genes significantly co enriched in melanocytes and iridophores illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis phosphoribosyl pyrophosphate thus constituting a guanine cycle. The purification procedure and expression analysis described here along with the accompanying transcriptome wide expression data provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 28 399s61,GSM1129619,,tissue:melanocytes|hpf,dm 77h 28 399s61,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013 based on NCBI's RefSeq build from 8/29/2012 current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129619,GSM1129619: dm 77h 28 399s61; Danio rerio; RNA Seq,GSM1129619 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129619,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_28_399s61_CACT.fq.bz2 dm_77h_28_399s62_CACT.fq.bz2,fastq fastq,731276158.0,3620179.0,GSM1129619 r1,0:101 1:101,A:173989639;C:199270484;G:188281318;T:169725601;N:9116,101,101,,,173989639,199270484,188281318,169725601,9116,SRX271957,SRS416253,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",2,0.84121,0.79968,0.02287,0.02141,0.89658,0.89051,0.63318,0.63061,101,101,B,B,biological fallback assumption,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36719,SRR835160,SRX271956,SRS416252,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes Iridophores and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology we developed a method to concomitantly purify melanocytes iridophores and retinal pigmented epithelium from zebrafish and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium as well as in melanocytes and iridophores. We found 214 genes co enriched in melanocytes and retinal pigmented epithelium indicating the shared functions of melanin producing cells. We found 62 genes significantly co enriched in melanocytes and iridophores illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis phosphoribosyl pyrophosphate thus constituting a guanine cycle. The purification procedure and expression analysis described here along with the accompanying transcriptome wide expression data provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 28 436,GSM1129618,,tissue:melanocytes|hpf,dm 77h 28 436,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013 based on NCBI's RefSeq build from 8/29/2012 current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129618,GSM1129618: dm 77h 28 436; Danio rerio; RNA Seq,GSM1129618 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129618,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_28_436_CACT.fq.bz2,fastq,214752678.0,5113159.0,GSM1129618 r1,0:42,A:50210098;C:62230664;G:49216487;T:52926929;N:168500,42,,,,50210098,62230664,49216487,52926929,168500,SRX271956,SRS416252,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.80956,,0.02412,,0.87024,,0.62475,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36720,SRR835159,SRX271955,SRS416251,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes Iridophores and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology we developed a method to concomitantly purify melanocytes iridophores and retinal pigmented epithelium from zebrafish and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium as well as in melanocytes and iridophores. We found 214 genes co enriched in melanocytes and retinal pigmented epithelium indicating the shared functions of melanin producing cells. We found 62 genes significantly co enriched in melanocytes and iridophores illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis phosphoribosyl pyrophosphate thus constituting a guanine cycle. The purification procedure and expression analysis described here along with the accompanying transcriptome wide expression data provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 28 351,GSM1129617,,tissue:melanocytes|hpf,dm 77h 28 351,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013 based on NCBI's RefSeq build from 8/29/2012 current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129617,GSM1129617: dm 77h 28 351; Danio rerio; RNA Seq,GSM1129617 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129617,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_28_351_CACT.fq.bz2,fastq,84584556.0,2349571.0,GSM1129617 r1,0:36,A:19843286;C:24761303;G:18855233;T:21124051;N:683,36,,,,19843286,24761303,18855233,21124051,683,SRX271955,SRS416251,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.75246,,0.02663,,0.87054,,0.59024,,36,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36721,SRR835158,SRX271954,SRS416250,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes Iridophores and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology we developed a method to concomitantly purify melanocytes iridophores and retinal pigmented epithelium from zebrafish and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium as well as in melanocytes and iridophores. We found 214 genes co enriched in melanocytes and retinal pigmented epithelium indicating the shared functions of melanin producing cells. We found 62 genes significantly co enriched in melanocytes and iridophores illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis phosphoribosyl pyrophosphate thus constituting a guanine cycle. The purification procedure and expression analysis described here along with the accompanying transcriptome wide expression data provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 27 436,GSM1129616,,tissue:melanocytes|hpf,dm 77h 27 436,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013 based on NCBI's RefSeq build from 8/29/2012 current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129616,GSM1129616: dm 77h 27 436; Danio rerio; RNA Seq,GSM1129616 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129616,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_27_436_TTAT.fq.bz2,fastq,194114466.0,4621773.0,GSM1129616 r1,0:42,A:44579558;C:47759429;G:44539735;T:57080409;N:155335,42,,,,44579558,47759429,44539735,57080409,155335,SRX271954,SRS416250,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.80787,,0.04019,,0.86263,,0.57373,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36722,SRR835157,SRX271953,SRS416249,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes Iridophores and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology we developed a method to concomitantly purify melanocytes iridophores and retinal pigmented epithelium from zebrafish and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium as well as in melanocytes and iridophores. We found 214 genes co enriched in melanocytes and retinal pigmented epithelium indicating the shared functions of melanin producing cells. We found 62 genes significantly co enriched in melanocytes and iridophores illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis phosphoribosyl pyrophosphate thus constituting a guanine cycle. The purification procedure and expression analysis described here along with the accompanying transcriptome wide expression data provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 27 351,GSM1129615,,tissue:melanocytes|hpf,dm 77h 27 351,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013 based on NCBI's RefSeq build from 8/29/2012 current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129615,GSM1129615: dm 77h 27 351; Danio rerio; RNA Seq,GSM1129615 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129615,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_27_351_TTAT.fq.bz2,fastq,113087304.0,3141314.0,GSM1129615 r1,0:36,A:26171623;C:27323878;G:24871328;T:34719586;N:889,36,,,,26171623,27323878,24871328,34719586,889,SRX271953,SRS416249,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.75375,,0.04337,,0.85878,,0.5311,,36,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36723,SRR835156,SRX271952,SRS416248,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes Iridophores and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology we developed a method to concomitantly purify melanocytes iridophores and retinal pigmented epithelium from zebrafish and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium as well as in melanocytes and iridophores. We found 214 genes co enriched in melanocytes and retinal pigmented epithelium indicating the shared functions of melanin producing cells. We found 62 genes significantly co enriched in melanocytes and iridophores illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis phosphoribosyl pyrophosphate thus constituting a guanine cycle. The purification procedure and expression analysis described here along with the accompanying transcriptome wide expression data provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 26 433,GSM1129614,,tissue:melanocytes|hpf,dm 77h 26 433,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013 based on NCBI's RefSeq build from 8/29/2012 current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129614,GSM1129614: dm 77h 26 433; Danio rerio; RNA Seq,GSM1129614 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129614,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_26_433_AGAT.fq.bz2,fastq,34005804.0,809662.0,GSM1129614 r1,0:42,A:8766789;C:8066807;G:8338043;T:8833768;N:397,42,,,,8766789,8066807,8338043,8833768,397,SRX271952,SRS416248,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.81802,,0.05684,,0.88787,,0.56756,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36724,SRR835155,SRX271951,SRS416247,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes Iridophores and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology we developed a method to concomitantly purify melanocytes iridophores and retinal pigmented epithelium from zebrafish and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium as well as in melanocytes and iridophores. We found 214 genes co enriched in melanocytes and retinal pigmented epithelium indicating the shared functions of melanin producing cells. We found 62 genes significantly co enriched in melanocytes and iridophores illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis phosphoribosyl pyrophosphate thus constituting a guanine cycle. The purification procedure and expression analysis described here along with the accompanying transcriptome wide expression data provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 26 351,GSM1129613,,tissue:melanocytes|hpf,dm 77h 26 351,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013 based on NCBI's RefSeq build from 8/29/2012 current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129613,GSM1129613: dm 77h 26 351; Danio rerio; RNA Seq,GSM1129613 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129613,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_26_351_AGAT.fq.bz2,fastq,101340432.0,2815012.0,GSM1129613 r1,0:36,A:26592120;C:23518331;G:24503636;T:26725495;N:850,36,,,,26592120,23518331,24503636,26725495,850,SRX271951,SRS416247,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.75158,,0.05787,,0.88986,,0.57167,,36,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36725,SRR835154,SRX271950,SRS416245,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes Iridophores and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology we developed a method to concomitantly purify melanocytes iridophores and retinal pigmented epithelium from zebrafish and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium as well as in melanocytes and iridophores. We found 214 genes co enriched in melanocytes and retinal pigmented epithelium indicating the shared functions of melanin producing cells. We found 62 genes significantly co enriched in melanocytes and iridophores illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis phosphoribosyl pyrophosphate thus constituting a guanine cycle. The purification procedure and expression analysis described here along with the accompanying transcriptome wide expression data provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 25 399s62,GSM1129612,,tissue:melanocytes|hpf,dm 77h 25 399s62,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013 based on NCBI's RefSeq build from 8/29/2012 current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129612,GSM1129612: dm 77h 25 399s62; Danio rerio; RNA Seq,GSM1129612 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129612,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,,,1715809008.0,8494104.0,GSM1129612 r1,0:101 1:101,A:405310561;C:444474389;G:467897835;T:398105078;N:21145,101,101,,,405310561,444474389,467897835,398105078,21145,SRX271950,SRS416245,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",2,0.66327,0.58026,0.07911,0.07288,0.91721,0.9083,0.56907,0.5363,101,101,B,B,biological fallback assumption,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36726,SRR835153,SRX271949,SRS416246,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes Iridophores and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology we developed a method to concomitantly purify melanocytes iridophores and retinal pigmented epithelium from zebrafish and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium as well as in melanocytes and iridophores. We found 214 genes co enriched in melanocytes and retinal pigmented epithelium indicating the shared functions of melanin producing cells. We found 62 genes significantly co enriched in melanocytes and iridophores illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis phosphoribosyl pyrophosphate thus constituting a guanine cycle. The purification procedure and expression analysis described here along with the accompanying transcriptome wide expression data provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 25 399s61,GSM1129611,,tissue:melanocytes|hpf,dm 77h 25 399s61,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013 based on NCBI's RefSeq build from 8/29/2012 current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129611,GSM1129611: dm 77h 25 399s61; Danio rerio; RNA Seq,GSM1129611 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129611,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_25_399s61_TCTT.fq.bz2 dm_77h_25_399s62_TCTT.fq.bz2,fastq fastq,1715809008.0,8494104.0,GSM1129611 r1,0:101 1:101,A:405310561;C:444474389;G:467897835;T:398105078;N:21145,101,101,,,405310561,444474389,467897835,398105078,21145,SRX271949,SRS416246,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",2,0.66325,0.58027,0.0792,0.07327,0.91741,0.90847,0.56374,0.53632,101,101,B,B,biological fallback assumption,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36727,SRR835152,SRX271948,SRS416244,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes Iridophores and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology we developed a method to concomitantly purify melanocytes iridophores and retinal pigmented epithelium from zebrafish and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium as well as in melanocytes and iridophores. We found 214 genes co enriched in melanocytes and retinal pigmented epithelium indicating the shared functions of melanin producing cells. We found 62 genes significantly co enriched in melanocytes and iridophores illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis phosphoribosyl pyrophosphate thus constituting a guanine cycle. The purification procedure and expression analysis described here along with the accompanying transcriptome wide expression data provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 25 433,GSM1129610,,tissue:melanocytes|hpf,dm 77h 25 433,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013 based on NCBI's RefSeq build from 8/29/2012 current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129610,GSM1129610: dm 77h 25 433; Danio rerio; RNA Seq,GSM1129610 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129610,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_25_433_TCTT.fq.bz2,fastq,489386604.0,11652062.0,GSM1129610 r1,0:42,A:99597908;C:134563195;G:119005661;T:136214210;N:5630,42,,,,99597908,134563195,119005661,136214210,5630,SRX271948,SRS416244,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.79072,,0.08396,,0.87497,,0.51318,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36728,SRR835151,SRX271947,SRS416243,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes Iridophores and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology we developed a method to concomitantly purify melanocytes iridophores and retinal pigmented epithelium from zebrafish and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium as well as in melanocytes and iridophores. We found 214 genes co enriched in melanocytes and retinal pigmented epithelium indicating the shared functions of melanin producing cells. We found 62 genes significantly co enriched in melanocytes and iridophores illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis phosphoribosyl pyrophosphate thus constituting a guanine cycle. The purification procedure and expression analysis described here along with the accompanying transcriptome wide expression data provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 25 351,GSM1129609,,tissue:melanocytes|hpf,dm 77h 25 351,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013 based on NCBI's RefSeq build from 8/29/2012 current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129609,GSM1129609: dm 77h 25 351; Danio rerio; RNA Seq,GSM1129609 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129609,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_25_351_TCTT.fq.bz2,fastq,98765532.0,2743487.0,GSM1129609 r1,0:36,A:19387910;C:27338385;G:23220852;T:28817575;N:810,36,,,,19387910,27338385,23220852,28817575,810,SRX271947,SRS416243,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.75369,,0.08166,,0.87667,,0.51755,,36,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36729,SRR835150,SRX271946,SRS416242,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes Iridophores and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology we developed a method to concomitantly purify melanocytes iridophores and retinal pigmented epithelium from zebrafish and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium as well as in melanocytes and iridophores. We found 214 genes co enriched in melanocytes and retinal pigmented epithelium indicating the shared functions of melanin producing cells. We found 62 genes significantly co enriched in melanocytes and iridophores illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis phosphoribosyl pyrophosphate thus constituting a guanine cycle. The purification procedure and expression analysis described here along with the accompanying transcriptome wide expression data provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 58h 45 440,GSM1129608,,tissue:melanocytes|hpf,dm 58h 45 440,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013 based on NCBI's RefSeq build from 8/29/2012 current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129608,GSM1129608: dm 58h 45 440; Danio rerio; RNA Seq,GSM1129608 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129608,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_58h_45_440.fq.bz2,fastq,203660982.0,4849071.0,GSM1129608 r1,0:42,A:46576136;C:50204145;G:47522343;T:59345685;N:12673,42,,,,46576136,50204145,47522343,59345685,12673,SRX271946,SRS416242,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.86133,,0.0978,,0.84565,,0.53118,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36730,SRR835149,SRX271945,SRS416241,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes Iridophores and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology we developed a method to concomitantly purify melanocytes iridophores and retinal pigmented epithelium from zebrafish and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium as well as in melanocytes and iridophores. We found 214 genes co enriched in melanocytes and retinal pigmented epithelium indicating the shared functions of melanin producing cells. We found 62 genes significantly co enriched in melanocytes and iridophores illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis phosphoribosyl pyrophosphate thus constituting a guanine cycle. The purification procedure and expression analysis described here along with the accompanying transcriptome wide expression data provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 58h 23 351,GSM1129607,,tissue:melanocytes|hpf,dm 58h 23 351,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013 based on NCBI's RefSeq build from 8/29/2012 current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129607,GSM1129607: dm 58h 23 351; Danio rerio; RNA Seq,GSM1129607 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236. Zebrafish were reared and bred according to standard protocols. The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor mitfavc7. This mutant facilitated the collection of RPE as mitfa is not required for RPE development in zebrafish. Melanocytes iridophores and RPE develop normally at 25°C in mitfavc7. When held at 32°C the neural crest derived melanocytes do not develop but RPE and iridophores develop normally. All melanocyte and iridophore samples were incubated at 25°C prior to collection. Fish were anesthetized with Tricaine rinsed with Ca Mg DPBS Sigma D8537 and immersed in 100mL TrypLE Express Invitrogen 12604039 per 1000 fish. Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes followed by trituration with a Pasteur pipette to remove eyes from larva. post separation of eyes and larva each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr. Dissociated cells were filtered through a 120uM screen into 50 mL tubes. Remaining intact tissue was triturated 10 20 times and again filtered through a 120uM screen into the dissociated cells. Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C then resuspended in 1mL cold isotonic Percoll Sigma P1644 by gentle pipetting. Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll. Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation. Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS and placed onto preformed Percoll density gradients. Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R. Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C. Following centrifugation overlying Percoll was aspirated leaving the final 100µL containing the pigment cell pellet. Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS and kept on ice until mRNA extraction or FACS. FACS We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS. Following resuspension in 500µL cold DPBS with 2% FCS the enriched cell populations were screened through a 30uM cell filter Partec 04 0042 2316. Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz. Cells were illuminated using a 488 nm laser. Cells were gated on two attributes to separate cells from each other and from cellular debris. Cellular debris was detected using forward and side scatter selecting against the smallest particles 1µm or less. Cells were sorted based on detection using 510 530nm and 575 595nm filters corresponding to FL1 and FL2 in Figure 1D respectively. When excited by the 488 nm laser the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant. Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser and cluster at the lower left of the FACS plot. Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows. For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions. Following mRNA elution from the Dynabeads first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview. A universal primer sequence was also added to the three prime end of the first strand by template switching allowing for PCR amplification of the resultant cDNA [43 44]. Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa PCR cycle: 95C for 1 minute followed by 20 cycles of 98C for 25 seconds 60C for 1 minute 68C for 20 minutes cDNA was digested with AluI and RsaI restriction enzymes NEB. Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation. Since this reduced representation strategy might miss short cDNAs that lack both restriction sites we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers. This allows for the inclusion of short cDNAs in our libraries. Standard Illumina library preparations followed performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu. In brief a single A was added to the 3’ end of each strand Y adapters ligated and library enrichment PCR performed followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length. Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45]. No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment. Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129607,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_58h_23_351_GAAT.fq.bz2,fastq,106123464.0,2947874.0,GSM1129607 r1,0:36,A:26495114;C:26882652;G:25518440;T:27226454;N:804,36,,,,26495114,26882652,25518440,27226454,804,SRX271945,SRS416241,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.78561,,0.06331,,0.85107,,0.46073,,36,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
38116,SRR1551799,SRX681419,SRS685080,SRP045504,PRJNA258223,Danio rerio strain:TL Transcriptome or Gene expression,PRJNA258223,Other,Transcriptome profiling of keratocytes derived from 2dpf and 4dpf embryos,,,,keratocytes from 4dpf embryos,keratocytes from 4dpf embryos,,breed:TL|strain:TL|age:4dpf|biomaterial provider:Julie Theriot Stanford University|sex:not collected|tissue:keratocytes skin|cell type:keratocyte|dev stage:4dpf|BioSampleModel:Model organism or animal,,,,,,,,,transcriptome from 4dpf keratocytes replicate 3,4dpf 3,4dpf 3,RNA was extracted from approximately 1 x 105 keratocytes per sample using Trizol Invitrogen. Three biological replicates were collected for each developmental stage. Ribosomal RNA was depleted using the Ribo Zero magnetic kit Epicentre. The remaining mRNA was then fragmented with 8 minutes incubation in 50mM sodium carbonate/bicarbonate 1mM EDTA pH 9.2 at 95 degrees. To prepare cDNA first strand synthesis was performed with Superscript III Invitrogen using random hexamer priming and second strand synthesis was performed with DNA Polymerase I NEB. Illumina libraries were prepared from the cDNA in an automated fashion using the Illumina TruSeq sample prep kit with TruSeq adapters on a SPRIworks System I Beckman Coulter. Sequencing reactions were performed on an Illumina Genome Analyzer IIX according to manufacturer’s instructions to generate 40nt single ended reads.,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,400Application ReadForward1,SRP045504,,,lane4_4dpf707.fastq,fastq,1001179200.0,25029480.0,4dpf 3,0:40,A:253770714;C:242097985;G:244642013;T:260557997;N:110491,40,,,,253770714,242097985,244642013,260557997,110491,SRX681419,SRS685080,SRA179326,Stanford University|Julie Theriot,Stanford University,1,0.90546,,0.2101,,0.75185,,0.49242,,40,,B,,usable mapping rate,illumina,early_illumina,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2015-04-07,Larval,Larval,Skin,Surface Structure
38117,SRR1551798,SRX681418,SRS685080,SRP045504,PRJNA258223,Danio rerio strain:TL Transcriptome or Gene expression,PRJNA258223,Other,Transcriptome profiling of keratocytes derived from 2dpf and 4dpf embryos,,,,keratocytes from 4dpf embryos,keratocytes from 4dpf embryos,,breed:TL|strain:TL|age:4dpf|biomaterial provider:Julie Theriot Stanford University|sex:not collected|tissue:keratocytes skin|cell type:keratocyte|dev stage:4dpf|BioSampleModel:Model organism or animal,,,,,,,,,Transcriptome from 4dpf keratocytes replicate 2,4dpf 2,4dpf 2,RNA was extracted from approximately 1 x 105 keratocytes per sample using Trizol Invitrogen. Three biological replicates were collected for each developmental stage. Ribosomal RNA was depleted using the Ribo Zero magnetic kit Epicentre. The remaining mRNA was then fragmented with 8 minutes incubation in 50mM sodium carbonate/bicarbonate 1mM EDTA pH 9.2 at 95 degrees. To prepare cDNA first strand synthesis was performed with Superscript III Invitrogen using random hexamer priming and second strand synthesis was performed with DNA Polymerase I NEB. Illumina libraries were prepared from the cDNA in an automated fashion using the Illumina TruSeq sample prep kit with TruSeq adapters on a SPRIworks System I Beckman Coulter. Sequencing reactions were performed on an Illumina Genome Analyzer IIX according to manufacturer’s instructions to generate 40nt single ended reads.,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,400Application ReadForward1,SRP045504,,,lane7_4col.fastq,fastq,1700157430.0,44740985.0,4dpf 2,0:38,A:394789834;C:418821057;G:504852317;T:369111176;N:12583046,38,,,,394789834,418821057,504852317,369111176,12583046,SRX681418,SRS685080,SRA179326,Stanford University|Julie Theriot,Stanford University,1,0.68441,,0.12529,,0.8338,,0.45494,,38,,B,,usable mapping rate,illumina,early_illumina,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2014-08-14,Larval,Larval,Skin,Surface Structure
38118,SRR1551797,SRX681417,SRS685080,SRP045504,PRJNA258223,Danio rerio strain:TL Transcriptome or Gene expression,PRJNA258223,Other,Transcriptome profiling of keratocytes derived from 2dpf and 4dpf embryos,,,,keratocytes from 4dpf embryos,keratocytes from 4dpf embryos,,breed:TL|strain:TL|age:4dpf|biomaterial provider:Julie Theriot Stanford University|sex:not collected|tissue:keratocytes skin|cell type:keratocyte|dev stage:4dpf|BioSampleModel:Model organism or animal,,,,,,,,,4dpf keratocyte transcriptome replicate 1,4dpf 1,4dpf 1,RNA was extracted from approximately 1 x 105 keratocytes per sample using Trizol Invitrogen. Three biological replicates were collected for each developmental stage. Ribosomal RNA was depleted using the Ribo Zero magnetic kit Epicentre. The remaining mRNA was then fragmented with 8 minutes incubation in 50mM sodium carbonate/bicarbonate 1mM EDTA pH 9.2 at 95 degrees. To prepare cDNA first strand synthesis was performed with Superscript III Invitrogen using random hexamer priming and second strand synthesis was performed with DNA Polymerase I NEB. Illumina libraries were prepared from the cDNA in an automated fashion using the Illumina TruSeq sample prep kit with TruSeq adapters on a SPRIworks System I Beckman Coulter. Sequencing reactions were performed on an Illumina Genome Analyzer IIX according to manufacturer’s instructions to generate 40nt single ended reads.,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,470Application ReadForward1,SRP045504,,,lane7_1_4dpf.fastq,fastq,762138229.0,16215707.0,4dpf 1,0:47,A:183899711;C:180347476;G:190086042;T:189460942;N:18344058,47,,,,183899711,180347476,190086042,189460942,18344058,SRX681417,SRS685080,SRA179326,Stanford University|Julie Theriot,Stanford University,1,0.85884,,0.2135,,0.80077,,0.50539,,47,,B,,usable mapping rate,illumina,early_illumina,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2014-08-15,Larval,Larval,Skin,Surface Structure
38119,SRR1551796,SRX681416,SRS685079,SRP045504,PRJNA258223,Danio rerio strain:TL Transcriptome or Gene expression,PRJNA258223,Other,Transcriptome profiling of keratocytes derived from 2dpf and 4dpf embryos,,,,Transcriptome from 2dpf keratocytes,keratocytes from 2dpf embryos,,breed:TL|strain:TL|age:2dpf|biomaterial provider:Julie Theriot Stanford University|sex:not collected|tissue:keratocytes skin|cell type:keratocyte|dev stage:2dpf|BioSampleModel:Model organism or animal,,,,,,,,,Transcriptome from 2dpf keratocytes replicate 3,2dpf 3,2dpf 3,RNA was extracted from approximately 1 x 105 keratocytes per sample using Trizol Invitrogen. Three biological replicates were collected for each developmental stage. Ribosomal RNA was depleted using the Ribo Zero magnetic kit Epicentre. The remaining mRNA was then fragmented with 8 minutes incubation in 50mM sodium carbonate/bicarbonate 1mM EDTA pH 9.2 at 95 degrees. To prepare cDNA first strand synthesis was performed with Superscript III Invitrogen using random hexamer priming and second strand synthesis was performed with DNA Polymerase I NEB. Illumina libraries were prepared from the cDNA in an automated fashion using the Illumina TruSeq sample prep kit with TruSeq adapters on a SPRIworks System I Beckman Coulter. Sequencing reactions were performed on an Illumina Genome Analyzer IIX according to manufacturer’s instructions to generate 40nt single ended reads.,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,400Application ReadForward1,SRP045504,,,lane3_2dpf629.fastq,fastq,551787520.0,13794688.0,2dpf 3,0:40,A:138660463;C:132605577;G:134640312;T:145852714;N:28454,40,,,,138660463,132605577,134640312,145852714,28454,SRX681416,SRS685079,SRA179323,Stanford University|Julie Theriot,Stanford University,1,0.91375,,0.21598,,0.75317,,0.47881,,40,,B,,usable mapping rate,illumina,early_illumina,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2014-08-14,Hatching,Embryo,Skin,Surface Structure
38120,SRR1551795,SRX681415,SRS685079,SRP045504,PRJNA258223,Danio rerio strain:TL Transcriptome or Gene expression,PRJNA258223,Other,Transcriptome profiling of keratocytes derived from 2dpf and 4dpf embryos,,,,Transcriptome from 2dpf keratocytes,keratocytes from 2dpf embryos,,breed:TL|strain:TL|age:2dpf|biomaterial provider:Julie Theriot Stanford University|sex:not collected|tissue:keratocytes skin|cell type:keratocyte|dev stage:2dpf|BioSampleModel:Model organism or animal,,,,,,,,,Transcriptome from 2dpf keratocytes,2dpf 2,2dpf 2,RNA was extracted from approximately 1 x 105 keratocytes per sample using Trizol Invitrogen. Three biological replicates were collected for each developmental stage. Ribosomal RNA was depleted using the Ribo Zero magnetic kit Epicentre. The remaining mRNA was then fragmented with 8 minutes incubation in 50mM sodium carbonate/bicarbonate 1mM EDTA pH 9.2 at 95 degrees. To prepare cDNA first strand synthesis was performed with Superscript III Invitrogen using random hexamer priming and second strand synthesis was performed with DNA Polymerase I NEB. Illumina libraries were prepared from the cDNA in an automated fashion using the Illumina TruSeq sample prep kit with TruSeq adapters on a SPRIworks System I Beckman Coulter. Sequencing reactions were performed on an Illumina Genome Analyzer IIX according to manufacturer’s instructions to generate 40nt single ended reads.,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,400Application ReadForward1,SRP045504,,,lane1_2dpf.fastq,fastq,796874112.0,22135392.0,2dpf 2,0:36,A:209956517;C:184659684;G:185142987;T:216055902;N:1059022,36,,,,209956517,184659684,185142987,216055902,1059022,SRX681415,SRS685079,SRA179323,Stanford University|Julie Theriot,Stanford University,1,0.89178,,0.17837,,0.7609,,0.49774,,36,,B,,usable mapping rate,illumina,early_illumina,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2015-04-07,Hatching,Embryo,Skin,Surface Structure
38121,SRR1551782,SRX681402,SRS685079,SRP045504,PRJNA258223,Danio rerio strain:TL Transcriptome or Gene expression,PRJNA258223,Other,Transcriptome profiling of keratocytes derived from 2dpf and 4dpf embryos,,,,Transcriptome from 2dpf keratocytes,keratocytes from 2dpf embryos,,breed:TL|strain:TL|age:2dpf|biomaterial provider:Julie Theriot Stanford University|sex:not collected|tissue:keratocytes skin|cell type:keratocyte|dev stage:2dpf|BioSampleModel:Model organism or animal,,,,,,,,,2dpf keratocytes replicate 1,2dpf 1,2dpf 1,RNA was extracted from approximately 1 x 105 keratocytes per sample using Trizol Invitrogen. Three biological replicates were collected for each developmental stage. Ribosomal RNA was depleted using the Ribo Zero magnetic kit Epicentre. The remaining mRNA was then fragmented with 8 minutes incubation in 50mM sodium carbonate/bicarbonate 1mM EDTA pH 9.2 at 95 degrees. To prepare cDNA first strand synthesis was performed with Superscript III Invitrogen using random hexamer priming and second strand synthesis was performed with DNA Polymerase I NEB. Illumina libraries were prepared from the cDNA in an automated fashion using the Illumina TruSeq sample prep kit with TruSeq adapters on a SPRIworks System I Beckman Coulter. Sequencing reactions were performed on an Illumina Genome Analyzer IIX according to manufacturer’s instructions to generate 40nt single ended reads.,,,RNA-Seq,TRANSCRIPTOMIC,PolyA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,400Application ReadForward1,SRP045504,,,lane6_2dpf.fastq,fastq,647944118.0,17051161.0,2dpf 1,0:38,A:145735255;C:167786338;G:186677806;T:141598169;N:6146550,38,,,,145735255,167786338,186677806,141598169,6146550,SRX681402,SRS685079,SRA179323,Stanford University|Julie Theriot,Stanford University,1,0.7695,,0.15816,,0.81491,,0.44972,,38,,B,,usable mapping rate,illumina,early_illumina,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2014-08-14,Hatching,Embryo,Skin,Surface Structure
38389,SRR1821850,SRX893451,SRS859516,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin208 36m 3.75nM roten1 8w rep6,GSM1620997,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,skin208 36m 3.75nM roten1 8w rep6,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,GSM1620997,GSM1620997: skin208 36m 3.75nM roten1 8w rep6; Danio rerio; RNA Seq,GSM1620997,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620997,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,208_skin.fastq.gz,fastq,1960697700.0,39213954.0,GSM1620997 r1,0:50,A:532792887;C:452528560;G:441240826;T:533522462;N:612965,50,,,,532792887,452528560,441240826,533522462,612965,SRX893451,SRS859516,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92216,,0.09326,,0.72147,,0.49795,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38390,SRR1821849,SRX893450,SRS859517,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin207 36m 3.75nM roten1 8w rep5,GSM1620996,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,skin207 36m 3.75nM roten1 8w rep5,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,GSM1620996,GSM1620996: skin207 36m 3.75nM roten1 8w rep5; Danio rerio; RNA Seq,GSM1620996,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620996,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,207_skin.fastq.gz,fastq,1644999850.0,32899997.0,GSM1620996 r1,0:50,A:444439104;C:381379643;G:371782441;T:447032625;N:366037,50,,,,444439104,381379643,371782441,447032625,366037,SRX893450,SRS859517,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.90733,,0.0971,,0.71518,,0.48175,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38391,SRR1821848,SRX893449,SRS859518,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin206 36m 3.75nM roten1 8w rep4,GSM1620995,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,skin206 36m 3.75nM roten1 8w rep4,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,GSM1620995,GSM1620995: skin206 36m 3.75nM roten1 8w rep4; Danio rerio; RNA Seq,GSM1620995,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620995,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,206_skin.fastq.gz,fastq,2333175800.0,46663516.0,GSM1620995 r1,0:50,A:629018600;C:541489412;G:532198591;T:629985712;N:483485,50,,,,629018600,541489412,532198591,629985712,483485,SRX893449,SRS859518,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91718,,0.10454,,0.70974,,0.47753,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38392,SRR1821847,SRX893448,SRS859519,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin205 36m 3.75nM roten1 8w rep3,GSM1620994,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,skin205 36m 3.75nM roten1 8w rep3,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,GSM1620994,GSM1620994: skin205 36m 3.75nM roten1 8w rep3; Danio rerio; RNA Seq,GSM1620994,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620994,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,205_skin.fastq.gz,fastq,1885120700.0,37702414.0,GSM1620994 r1,0:50,A:509247143;C:431669478;G:422352918;T:511569209;N:10281952,50,,,,509247143,431669478,422352918,511569209,10281952,SRX893448,SRS859519,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.90066,,0.107,,0.71415,,0.48729,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38393,SRR1821846,SRX893447,SRS859520,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin204 36m 3.75nM roten1 8w rep2,GSM1620993,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,skin204 36m 3.75nM roten1 8w rep2,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,GSM1620993,GSM1620993: skin204 36m 3.75nM roten1 8w rep2; Danio rerio; RNA Seq,GSM1620993,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620993,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,204_skin.fastq.gz,fastq,2039214400.0,40784288.0,GSM1620993 r1,0:50,A:554630438;C:468936669;G:460030131;T:555276071;N:341091,50,,,,554630438,468936669,460030131,555276071,341091,SRX893447,SRS859520,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91261,,0.11181,,0.70832,,0.4889,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38394,SRR1821845,SRX893446,SRS859521,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin203 36m 3.75nM roten1 8w rep1,GSM1620992,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,skin203 36m 3.75nM roten1 8w rep1,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,GSM1620992,GSM1620992: skin203 36m 3.75nM roten1 8w rep1; Danio rerio; RNA Seq,GSM1620992,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620992,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,203_skin.fastq.gz,fastq,2169512850.0,43390257.0,GSM1620992 r1,0:50,A:583644936;C:503133232;G:492417551;T:585152349;N:5164782,50,,,,583644936,503133232,492417551,585152349,5164782,SRX893446,SRS859521,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91263,,0.1004,,0.71376,,0.48566,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38395,SRR1821844,SRX893445,SRS859522,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin172 36m 3.75nM roten1 3w rep5,GSM1620991,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:3.75nM roten1|duration:3 weeks,skin172 36m 3.75nM roten1 3w rep5,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:3.75nM roten1|duration:3 weeks,GSM1620991,GSM1620991: skin172 36m 3.75nM roten1 3w rep5; Danio rerio; RNA Seq,GSM1620991,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620991,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,172_skin.fastq.gz,fastq,1922415800.0,38448316.0,GSM1620991 r1,0:50,A:517025440;C:447202733;G:438389966;T:519578929;N:218732,50,,,,517025440,447202733,438389966,519578929,218732,SRX893445,SRS859522,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92509,,0.08635,,0.71747,,0.48682,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38396,SRR1821843,SRX893444,SRS859523,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin171 36m 3.75nM roten1 3w rep4,GSM1620990,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:3.75nM roten1|duration:3 weeks,skin171 36m 3.75nM roten1 3w rep4,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:3.75nM roten1|duration:3 weeks,GSM1620990,GSM1620990: skin171 36m 3.75nM roten1 3w rep4; Danio rerio; RNA Seq,GSM1620990,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620990,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,171_skin.fastq.gz,fastq,1795802450.0,35916049.0,GSM1620990 r1,0:50,A:482833890;C:415521773;G:409072097;T:485286706;N:3087984,50,,,,482833890,415521773,409072097,485286706,3087984,SRX893444,SRS859523,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91548,,0.08856,,0.71153,,0.48407,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38397,SRR1821842,SRX893443,SRS859524,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin170 36m 3.75nM roten1 3w rep3,GSM1620989,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:3.75nM roten1|duration:3 weeks,skin170 36m 3.75nM roten1 3w rep3,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:3.75nM roten1|duration:3 weeks,GSM1620989,GSM1620989: skin170 36m 3.75nM roten1 3w rep3; Danio rerio; RNA Seq,GSM1620989,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620989,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,170_skin.fastq.gz,fastq,1844753700.0,36895074.0,GSM1620989 r1,0:50,A:496864420;C:428934827;G:420432768;T:498397505;N:124180,50,,,,496864420,428934827,420432768,498397505,124180,SRX893443,SRS859524,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92475,,0.08478,,0.71123,,0.5082,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38398,SRR1821841,SRX893442,SRS859525,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin169 36m 3.75nM roten1 3w rep2,GSM1620988,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:3.75nM roten1|duration:3 weeks,skin169 36m 3.75nM roten1 3w rep2,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:3.75nM roten1|duration:3 weeks,GSM1620988,GSM1620988: skin169 36m 3.75nM roten1 3w rep2; Danio rerio; RNA Seq,GSM1620988,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620988,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,169_skin.fastq.gz,fastq,1832979850.0,36659597.0,GSM1620988 r1,0:50,A:492012429;C:429700408;G:417026859;T:494015583;N:224571,50,,,,492012429,429700408,417026859,494015583,224571,SRX893442,SRS859525,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93738,,0.07334,,0.71936,,0.5123,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38399,SRR1821840,SRX893441,SRS859526,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin168 36m 3.75nM roten1 3w rep1,GSM1620987,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:3.75nM roten1|duration:3 weeks,skin168 36m 3.75nM roten1 3w rep1,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:3.75nM roten1|duration:3 weeks,GSM1620987,GSM1620987: skin168 36m 3.75nM roten1 3w rep1; Danio rerio; RNA Seq,GSM1620987,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620987,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,168_skin.fastq.gz,fastq,1811602600.0,36232052.0,GSM1620987 r1,0:50,A:482632140;C:422445561;G:412836586;T:485392445;N:8295868,50,,,,482632140,422445561,412836586,485392445,8295868,SRX893441,SRS859526,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.9185,,0.07569,,0.71368,,0.49409,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38400,SRR1821839,SRX893440,SRS859527,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin188 36m control 8w rep4,GSM1620986,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:n1|duration:8 weeks,skin188 36m control 8w rep4,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:n1|duration:8 weeks,GSM1620986,GSM1620986: skin188 36m control 8w rep4; Danio rerio; RNA Seq,GSM1620986,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620986,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,188_skin.fastq.gz,fastq,1797310350.0,35946207.0,GSM1620986 r1,0:50,A:486641633;C:413978398;G:404993329;T:489015902;N:2681088,50,,,,486641633,413978398,404993329,489015902,2681088,SRX893440,SRS859527,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91594,,0.09158,,0.72082,,0.51585,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38401,SRR1821838,SRX893439,SRS859528,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin187 36m control 8w rep3,GSM1620985,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:n1|duration:8 weeks,skin187 36m control 8w rep3,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:n1|duration:8 weeks,GSM1620985,GSM1620985: skin187 36m control 8w rep3; Danio rerio; RNA Seq,GSM1620985,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620985,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,187_skin.fastq.gz,fastq,1951002950.0,39020059.0,GSM1620985 r1,0:50,A:520701869;C:457907686;G:446812256;T:524196019;N:1385120,50,,,,520701869,457907686,446812256,524196019,1385120,SRX893439,SRS859528,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.9211,,0.07622,,0.71145,,0.46944,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38402,SRR1821837,SRX893438,SRS859529,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin186 36m control 8w rep2,GSM1620984,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:n1|duration:8 weeks,skin186 36m control 8w rep2,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:n1|duration:8 weeks,GSM1620984,GSM1620984: skin186 36m control 8w rep2; Danio rerio; RNA Seq,GSM1620984,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620984,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,186_skin.fastq.gz,fastq,2016117600.0,40322352.0,GSM1620984 r1,0:50,A:545084574;C:465634059;G:454024180;T:546321124;N:5053663,50,,,,545084574,465634059,454024180,546321124,5053663,SRX893438,SRS859529,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91606,,0.09659,,0.71246,,0.52973,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38403,SRR1821836,SRX893437,SRS859530,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin185 36m control 8w rep1,GSM1620983,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:n1|duration:8 weeks,skin185 36m control 8w rep1,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:n1|duration:8 weeks,GSM1620983,GSM1620983: skin185 36m control 8w rep1; Danio rerio; RNA Seq,GSM1620983,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620983,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,185_skin.fastq.gz,fastq,1862747450.0,37254949.0,GSM1620983 r1,0:50,A:506321471;C:429322966;G:419158880;T:507760233;N:183900,50,,,,506321471,429322966,419158880,507760233,183900,SRX893437,SRS859530,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.9187,,0.09926,,0.70755,,0.51593,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38404,SRR1821835,SRX893436,SRS859531,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin162 36m control 3w rep3,GSM1620982,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:n1|duration:3 weeks,skin162 36m control 3w rep3,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:n1|duration:3 weeks,GSM1620982,GSM1620982: skin162 36m control 3w rep3; Danio rerio; RNA Seq,GSM1620982,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620982,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,162_skin.fastq.gz,fastq,2058728600.0,41174572.0,GSM1620982 r1,0:50,A:545731040;C:487775330;G:479420254;T:545686109;N:115867,50,,,,545731040,487775330,479420254,545686109,115867,SRX893436,SRS859531,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93508,,0.07365,,0.70688,,0.4827,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38405,SRR1821834,SRX893435,SRS859532,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin161 36m control 3w rep2,GSM1620981,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:n1|duration:3 weeks,skin161 36m control 3w rep2,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:n1|duration:3 weeks,GSM1620981,GSM1620981: skin161 36m control 3w rep2; Danio rerio; RNA Seq,GSM1620981,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620981,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,161_skin.fastq.gz,fastq,1897142450.0,37942849.0,GSM1620981 r1,0:50,A:508043218;C:440251400;G:428510637;T:511046395;N:9290800,50,,,,508043218,440251400,428510637,511046395,9290800,SRX893435,SRS859532,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.90644,,0.0826,,0.71358,,0.49573,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38406,SRR1821833,SRX893434,SRS859533,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin160 36m control 3w rep1,GSM1620980,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:n1|duration:3 weeks,skin160 36m control 3w rep1,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:n1|duration:3 weeks,GSM1620980,GSM1620980: skin160 36m control 3w rep1; Danio rerio; RNA Seq,GSM1620980,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620980,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,160_skin.fastq.gz,fastq,1829692650.0,36593853.0,GSM1620980 r1,0:50,A:493906226;C:425214476;G:414775189;T:495470406;N:326353,50,,,,493906226,425214476,414775189,495470406,326353,SRX893434,SRS859533,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92036,,0.09412,,0.71484,,0.48928,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38407,SRR1821832,SRX893433,SRS859534,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin184 12m control 8w rep3,GSM1620979,,source name:total RNA extracted from skin|tissue:skin|age:12 month|treatment:n1|duration:8 weeks,skin184 12m control 8w rep3,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:12 month|treatment:n1|duration:8 weeks,GSM1620979,GSM1620979: skin184 12m control 8w rep3; Danio rerio; RNA Seq,GSM1620979,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620979,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,184_skin.fastq.gz,fastq,1888865600.0,37777312.0,GSM1620979 r1,0:50,A:510384459;C:436312051;G:428261723;T:512241273;N:1666094,50,,,,510384459,436312051,428261723,512241273,1666094,SRX893433,SRS859534,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91542,,0.0997,,0.70082,,0.49633,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38408,SRR1821831,SRX893432,SRS859535,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin183 12m control 8w rep2,GSM1620978,,source name:total RNA extracted from skin|tissue:skin|age:12 month|treatment:n1|duration:8 weeks,skin183 12m control 8w rep2,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:12 month|treatment:n1|duration:8 weeks,GSM1620978,GSM1620978: skin183 12m control 8w rep2; Danio rerio; RNA Seq,GSM1620978,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620978,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,183_skin.fastq.gz,fastq,2011969000.0,40239380.0,GSM1620978 r1,0:50,A:541080523;C:469282719;G:455255702;T:544440274;N:1909782,50,,,,541080523,469282719,455255702,544440274,1909782,SRX893432,SRS859535,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92404,,0.104,,0.70619,,0.47963,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38409,SRR1821830,SRX893431,SRS859536,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin182 12m control 8w rep1,GSM1620977,,source name:total RNA extracted from skin|tissue:skin|age:12 month|treatment:n1|duration:8 weeks,skin182 12m control 8w rep1,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:12 month|treatment:n1|duration:8 weeks,GSM1620977,GSM1620977: skin182 12m control 8w rep1; Danio rerio; RNA Seq,GSM1620977,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620977,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,182_skin.fastq.gz,fastq,1974072600.0,39481452.0,GSM1620977 r1,0:50,A:532294515;C:457485293;G:447416004;T:532212696;N:4664092,50,,,,532294515,457485293,447416004,532212696,4664092,SRX893431,SRS859536,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91699,,0.10118,,0.71163,,0.49086,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38410,SRR1821829,SRX893430,SRS859537,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin159 12m control 3w rep3,GSM1620976,,source name:total RNA extracted from skin|tissue:skin|age:12 month|treatment:n1|duration:3 weeks,skin159 12m control 3w rep3,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:12 month|treatment:n1|duration:3 weeks,GSM1620976,GSM1620976: skin159 12m control 3w rep3; Danio rerio; RNA Seq,GSM1620976,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620976,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,159_skin.fastq.gz,fastq,1887853800.0,37757076.0,GSM1620976 r1,0:50,A:510759018;C:434865500;G:426519929;T:512499569;N:3209784,50,,,,510759018,434865500,426519929,512499569,3209784,SRX893430,SRS859537,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91654,,0.1023,,0.70607,,0.49388,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38411,SRR1821828,SRX893429,SRS859538,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin158 12m control 3w rep2,GSM1620975,,source name:total RNA extracted from skin|tissue:skin|age:12 month|treatment:n1|duration:3 weeks,skin158 12m control 3w rep2,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:12 month|treatment:n1|duration:3 weeks,GSM1620975,GSM1620975: skin158 12m control 3w rep2; Danio rerio; RNA Seq,GSM1620975,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620975,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,158_skin.fastq.gz,fastq,1818559300.0,36371186.0,GSM1620975 r1,0:50,A:487547586;C:425039144;G:415674047;T:490148749;N:149774,50,,,,487547586,425039144,415674047,490148749,149774,SRX893429,SRS859538,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93033,,0.09871,,0.66103,,0.48246,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38412,SRR1821827,SRX893428,SRS859539,SRP055573,PRJNA276667,RNA seq of zebrafish brain liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain liver skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin157 12m control 3w rep1,GSM1620974,,source name:total RNA extracted from skin|tissue:skin|age:12 month|treatment:n1|duration:3 weeks,skin157 12m control 3w rep1,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and 8 weeks respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:12 month|treatment:n1|duration:3 weeks,GSM1620974,GSM1620974: skin157 12m control 3w rep1; Danio rerio; RNA Seq,GSM1620974,,1,Total RNA was isolated using QIAzol Qiagen Hilden Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab Erlangen Germany and stored at 80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA USA following the manufacturer's instruction.,GEO Accession:GSM1620974,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,157_skin.fastq.gz,fastq,1855321100.0,37106422.0,GSM1620974 r1,0:50,A:495304825;C:434601244;G:425046390;T:498975673;N:1392968,50,,,,495304825,434601244,425046390,498975673,1392968,SRX893428,SRS859539,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91768,,0.09102,,0.7012,,0.49299,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
40001,SRR2751044,SRX1362114,SRS1125416,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain liver skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6 12 24 36 42 month; 3 tissues brain liver skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 111,GSM1915550,,source name:total RNA extracted from skin|strain:AB JxTu Tgwt1a:GFP|age:42 month|age category:old 2|tissue:skin,NH FLI skin 111,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account: tophat2 p 24 o OUTPUTDIR g 1 no coverage search transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count quiet stranded=no idattr=gene id mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al. 2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgwt1a:GFP|age:42 month|age category:old 2|tissue:skin,GSM1915550,GSM1915550: NH FLI skin 111; Danio rerio; RNA Seq,GSM1915550,,1,Total RNA from was extracted as described Baumgart et al. 2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915550,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_42m_DR111.fq.gz,fastq,3159101450.0,63182029.0,GSM1915550 r1,0:50,A:848692326;C:737950616;G:722461308;T:849228405;N:768795,50,,,,848692326,737950616,722461308,849228405,768795,SRX1362114,SRS1125416,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.9327,,0.09435,,0.70737,,0.50049,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40002,SRR2751043,SRX1362113,SRS1125415,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain liver skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6 12 24 36 42 month; 3 tissues brain liver skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 109,GSM1915549,,source name:total RNA extracted from skin|strain:AB JxTu Tgwt1a:GFP|age:42 month|age category:old 2|tissue:skin,NH FLI skin 109,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account: tophat2 p 24 o OUTPUTDIR g 1 no coverage search transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count quiet stranded=no idattr=gene id mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al. 2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgwt1a:GFP|age:42 month|age category:old 2|tissue:skin,GSM1915549,GSM1915549: NH FLI skin 109; Danio rerio; RNA Seq,GSM1915549,,1,Total RNA from was extracted as described Baumgart et al. 2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915549,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_42m_DR109.fq.gz,fastq,2638118700.0,52762374.0,GSM1915549 r1,0:50,A:699567604;C:623885297;G:610871040;T:703405737;N:389022,50,,,,699567604,623885297,610871040,703405737,389022,SRX1362113,SRS1125415,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93453,,0.09144,,0.7191,,0.47437,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40003,SRR2751042,SRX1362112,SRS1125418,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain liver skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6 12 24 36 42 month; 3 tissues brain liver skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 108,GSM1915548,,source name:total RNA extracted from skin|strain:AB JxTu Tgwt1a:GFP|age:42 month|age category:old 2|tissue:skin,NH FLI skin 108,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account: tophat2 p 24 o OUTPUTDIR g 1 no coverage search transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count quiet stranded=no idattr=gene id mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al. 2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgwt1a:GFP|age:42 month|age category:old 2|tissue:skin,GSM1915548,GSM1915548: NH FLI skin 108; Danio rerio; RNA Seq,GSM1915548,,1,Total RNA from was extracted as described Baumgart et al. 2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915548,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_42m_DR108.fq.gz,fastq,2354900150.0,47098003.0,GSM1915548 r1,0:50,A:629252405;C:552452722;G:540951538;T:632145411;N:98074,50,,,,629252405,552452722,540951538,632145411,98074,SRX1362112,SRS1125418,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93959,,0.09384,,0.71764,,0.49807,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40004,SRR2751041,SRX1362111,SRS1125419,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain liver skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6 12 24 36 42 month; 3 tissues brain liver skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 107,GSM1915547,,source name:total RNA extracted from skin|strain:AB JxTu Tgwt1a:GFP|age:42 month|age category:old 2|tissue:skin,NH FLI skin 107,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account: tophat2 p 24 o OUTPUTDIR g 1 no coverage search transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count quiet stranded=no idattr=gene id mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al. 2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgwt1a:GFP|age:42 month|age category:old 2|tissue:skin,GSM1915547,GSM1915547: NH FLI skin 107; Danio rerio; RNA Seq,GSM1915547,,1,Total RNA from was extracted as described Baumgart et al. 2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915547,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_42m_DR107.fq.gz,fastq,2518777200.0,50375544.0,GSM1915547 r1,0:50,A:678501999;C:582249482;G:579224840;T:678502233;N:298646,50,,,,678501999,582249482,579224840,678502233,298646,SRX1362111,SRS1125419,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.9366,,0.10967,,0.71977,,0.4795,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40005,SRR2751040,SRX1362110,SRS1125417,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain liver skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6 12 24 36 42 month; 3 tissues brain liver skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 106,GSM1915546,,source name:total RNA extracted from skin|strain:AB JxTu Tgwt1a:GFP|age:42 month|age category:old 2|tissue:skin,NH FLI skin 106,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account: tophat2 p 24 o OUTPUTDIR g 1 no coverage search transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count quiet stranded=no idattr=gene id mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al. 2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgwt1a:GFP|age:42 month|age category:old 2|tissue:skin,GSM1915546,GSM1915546: NH FLI skin 106; Danio rerio; RNA Seq,GSM1915546,,1,Total RNA from was extracted as described Baumgart et al. 2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915546,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_42m_DR106.fq.gz,fastq,2372061450.0,47441229.0,GSM1915546 r1,0:50,A:644690580;C:546357177;G:533675326;T:646884182;N:454185,50,,,,644690580,546357177,533675326,646884182,454185,SRX1362110,SRS1125417,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92831,,0.10593,,0.70453,,0.49444,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40006,SRR2751039,SRX1362109,SRS1125433,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain liver skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6 12 24 36 42 month; 3 tissues brain liver skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 150,GSM1915545,,source name:total RNA extracted from skin|strain:AB JxTu Tgpu.1:GFP|age:36 month|age category:old 1|tissue:skin,NH FLI skin 150,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account: tophat2 p 24 o OUTPUTDIR g 1 no coverage search transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count quiet stranded=no idattr=gene id mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al. 2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgpu.1:GFP|age:36 month|age category:old 1|tissue:skin,GSM1915545,GSM1915545: NH FLI skin 150; Danio rerio; RNA Seq,GSM1915545,,1,Total RNA from was extracted as described Baumgart et al. 2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915545,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_36m_DR150.fq.gz,fastq,2518891400.0,50377828.0,GSM1915545 r1,0:50,A:674241151;C:589651596;G:577346091;T:677408422;N:244140,50,,,,674241151,589651596,577346091,677408422,244140,SRX1362109,SRS1125433,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92457,,0.09542,,0.72985,,0.47778,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40007,SRR2751038,SRX1362108,SRS1125421,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain liver skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6 12 24 36 42 month; 3 tissues brain liver skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 149,GSM1915544,,source name:total RNA extracted from skin|strain:AB JxTu Tgpu.1:GFP|age:36 month|age category:old 1|tissue:skin,NH FLI skin 149,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account: tophat2 p 24 o OUTPUTDIR g 1 no coverage search transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count quiet stranded=no idattr=gene id mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al. 2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgpu.1:GFP|age:36 month|age category:old 1|tissue:skin,GSM1915544,GSM1915544: NH FLI skin 149; Danio rerio; RNA Seq,GSM1915544,,1,Total RNA from was extracted as described Baumgart et al. 2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915544,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_36m_DR149.fq.gz,fastq,2379227850.0,47584557.0,GSM1915544 r1,0:50,A:637191017;C:555983414;G:544406685;T:641356487;N:290247,50,,,,637191017,555983414,544406685,641356487,290247,SRX1362108,SRS1125421,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92532,,0.09729,,0.72813,,0.46517,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40008,SRR2751037,SRX1362107,SRS1125420,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain liver skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6 12 24 36 42 month; 3 tissues brain liver skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 147,GSM1915543,,source name:total RNA extracted from skin|strain:AB JxTu Tgpu.1:GFP|age:36 month|age category:old 1|tissue:skin,NH FLI skin 147,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account: tophat2 p 24 o OUTPUTDIR g 1 no coverage search transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count quiet stranded=no idattr=gene id mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al. 2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgpu.1:GFP|age:36 month|age category:old 1|tissue:skin,GSM1915543,GSM1915543: NH FLI skin 147; Danio rerio; RNA Seq,GSM1915543,,1,Total RNA from was extracted as described Baumgart et al. 2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915543,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_36m_DR147.fq.gz,fastq,2551716800.0,51034336.0,GSM1915543 r1,0:50,A:678806838;C:599057712;G:596701555;T:677051257;N:99438,50,,,,678806838,599057712,596701555,677051257,99438,SRX1362107,SRS1125420,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92591,,0.09543,,0.72648,,0.45136,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure