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
25297,SRR25764099,SRX21486772,SRS18719072,SRP457109,PRJNA1009810,Live tracking of basal stem cells of the epidermis during growth  homeostasis  and injury response in zebrafish,GSE241757,Transcriptome Analysis,Basal stem cells of the epidermis continuously differentiate into keratinocytes and replenish themselves via self renewal to maintain skin homeostasis. Numerous studies have attempted to reveal how basal cells undergo differentiation or self renewal; however  this has been hampered by a lack of robust basal cell markers and analytical platforms that allow single cell tracking. Here  we report integrin b4 itgb4 is a useful marker for basal cell labelling  irrespective of the body region  stage  and regenerative status. We employed Cre loxP recombination in combination with live cell tracking of single basal clones in the caudal fin and investigated the embryonic origin and behaviour of basal cells during fish growth and homeostasis. Although most basal cells  including those in fins  became quiescent in the adult stage  genetic cell ablation showed that basal cells were reactivated to either self renew or differentiate  depending on the injured cell type. Our findings provide a platform for quantitative in vivo imaging of basal stem cells applicable at wider stages and under various conditions and reveal unanticipated basal cell dynamism in response to distinct wound signals. Overall design: To investigate whether itgb4+ epidermal basal precursors already possess a basal cell like identity immediately post fate segregation at 1 dpf dpf  we isolated itgb4+ basal precursors  krt4+ periderm cells from zebrafish embryos at 1 dpf  and adult itgb4+ basal cells from zebrafish caudal fin by fluorescence activated cell sorting. We then performed gene expression profiling analysis using data obtained from RNA seq of 3 different cells at 2 time points. Comparative gene expression profiling analysis of RNA seq data for embryonic basal cell precursors  embryonic periderm  and adult basal cells.,,pubmed:38265193,,Adult itgb4+ basal cells,GSM7734879,,tissue:epidermal cells|cell type:adult fin epidermal basal cells|genotype:Tgitgb4:mcherry|geo loc name:missing|collection date:missing,Adult itgb4+ basal cells,High quality reads were mapped using hisat2 ver. 2.2.1 Trimmed sequence reads were mapped to GRCz11 using hisat2. Read count extraxction and normalization were performed using featureCounts ver. 2.0.3 Assembly: GRCz11 Supplementary files format and content: tab delimited text file includes basic gene information gene id  length  Genebank id  RNA type  gene name  product  raw count  RPKM and TPM values.,epidermal cells,Different types of cells were sorted by the fluorescence activated cell sorter SH800S SONY.,The sorted cells were directly collected in a lysis buffer and used for RNA extraction using the RNeasy Plus Micro Kit Qiagen RNA libraries were prepared by MGIEasy™ WES rapid library preparation kit according to the manufacturer’s protocol.,Fish were maintained in a recirculating water system in a 14 h day/10 h night photoperiod at 28.5 °C. The wild type zebrafish Danio rerio strain used in this study was originally derived from the Tubingen strain and has been maintained in our facility for more than 10 years by inbreeding.,cell type:adult fin epidermal basal cells|genotype:Tgitgb4:mcherry,GSM7734879,GSM7734879: Adult itgb4+ basal cells; Danio rerio; RNA Seq,GSM7734879 r1,GSM7734879,1,The sorted cells were directly collected in a lysis buffer and used for RNA extraction using the RNeasy Plus Micro Kit Qiagen RNA libraries were prepared by MGIEasy™ WES rapid library preparation kit according to the manufacturer's protocol.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,DNBSEQ,DNBSEQ-G400,,SRP457109,,,Adult_itgb4mcherry_S230_R1_001.fastq.gz Adult_itgb4mcherry_S230_R2_001.fastq.gz,fastq fastq,5035430000.0,12588575.0,GSM7734879 r1,0:200 1:200,A:1339392170;C:1117195164;G:1254708952;T:1323174485;N:959229,200,200,,,1339392170,1117195164,1254708952,1323174485,959229,SRX21486772,SRS18719072,SRA1700438,"Kawakami Lab, School of Life Science and Technology, Tokyo Institute of Technology","Kawakami Lab, School of Life Science and Technology, Tokyo Institute of Technology",2,0.01543,0.02468,0.00098,0.00102,0.99318,0.99387,0.47878,0.31718,200,200,T,T,mates < 9% mapping rate,bgi,bgi,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Japan,2023-08-28,Adult,Adult,Skin,Surface Structure
25298,SRR25764100,SRX21486771,SRS18719071,SRP457109,PRJNA1009810,Live tracking of basal stem cells of the epidermis during growth  homeostasis  and injury response in zebrafish,GSE241757,Transcriptome Analysis,Basal stem cells of the epidermis continuously differentiate into keratinocytes and replenish themselves via self renewal to maintain skin homeostasis. Numerous studies have attempted to reveal how basal cells undergo differentiation or self renewal; however  this has been hampered by a lack of robust basal cell markers and analytical platforms that allow single cell tracking. Here  we report integrin b4 itgb4 is a useful marker for basal cell labelling  irrespective of the body region  stage  and regenerative status. We employed Cre loxP recombination in combination with live cell tracking of single basal clones in the caudal fin and investigated the embryonic origin and behaviour of basal cells during fish growth and homeostasis. Although most basal cells  including those in fins  became quiescent in the adult stage  genetic cell ablation showed that basal cells were reactivated to either self renew or differentiate  depending on the injured cell type. Our findings provide a platform for quantitative in vivo imaging of basal stem cells applicable at wider stages and under various conditions and reveal unanticipated basal cell dynamism in response to distinct wound signals. Overall design: To investigate whether itgb4+ epidermal basal precursors already possess a basal cell like identity immediately post fate segregation at 1 dpf dpf  we isolated itgb4+ basal precursors  krt4+ periderm cells from zebrafish embryos at 1 dpf  and adult itgb4+ basal cells from zebrafish caudal fin by fluorescence activated cell sorting. We then performed gene expression profiling analysis using data obtained from RNA seq of 3 different cells at 2 time points. Comparative gene expression profiling analysis of RNA seq data for embryonic basal cell precursors  embryonic periderm  and adult basal cells.,,pubmed:38265193,,1 dpf itgb4+ embryonic basal precursors rep2,GSM7734878,,tissue:epidermal cells|cell type:embryonic epidermal basal cells|genotype:Tgitgb4:mcherry|geo loc name:missing|collection date:missing,1 dpf itgb4+ embryonic basal precursors rep2,High quality reads were mapped using hisat2 ver. 2.2.1 Trimmed sequence reads were mapped to GRCz11 using hisat2. Read count extraxction and normalization were performed using featureCounts ver. 2.0.3 Assembly: GRCz11 Supplementary files format and content: tab delimited text file includes basic gene information gene id  length  Genebank id  RNA type  gene name  product  raw count  RPKM and TPM values.,epidermal cells,Different types of cells were sorted by the fluorescence activated cell sorter SH800S SONY.,The sorted cells were directly collected in a lysis buffer and used for RNA extraction using the RNeasy Plus Micro Kit Qiagen RNA libraries were prepared by MGIEasy™ WES rapid library preparation kit according to the manufacturer’s protocol.,Fish were maintained in a recirculating water system in a 14 h day/10 h night photoperiod at 28.5 °C. The wild type zebrafish Danio rerio strain used in this study was originally derived from the Tubingen strain and has been maintained in our facility for more than 10 years by inbreeding.,cell type:embryonic epidermal basal cells|genotype:Tgitgb4:mcherry,GSM7734878,GSM7734878: 1 dpf itgb4+ embryonic basal precursors rep2; Danio rerio; RNA Seq,GSM7734878 r1,GSM7734878,1,The sorted cells were directly collected in a lysis buffer and used for RNA extraction using the RNeasy Plus Micro Kit Qiagen RNA libraries were prepared by MGIEasy™ WES rapid library preparation kit according to the manufacturer's protocol.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,DNBSEQ,DNBSEQ-G400,,SRP457109,,,itgb4mcherry_2_S229_R1_001.fastq.gz itgb4mcherry_2_S229_R2_001.fastq.gz,fastq fastq,6040245600.0,15100614.0,GSM7734878 r1,0:200 1:200,A:1588346322;C:1356558310;G:1524614760;T:1569583583;N:1142625,200,200,,,1588346322,1356558310,1524614760,1569583583,1142625,SRX21486771,SRS18719071,SRA1700438,"Kawakami Lab, School of Life Science and Technology, Tokyo Institute of Technology","Kawakami Lab, School of Life Science and Technology, Tokyo Institute of Technology",2,0.0156,0.0191,0.00074,0.00093,0.99024,0.99088,0.47058,0.47838,200,200,T,T,mates < 9% mapping rate,bgi,bgi,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Japan,2023-08-28,Multi-stage,Multi-stage,Skin,Surface Structure
25299,SRR25764101,SRX21486770,SRS18719070,SRP457109,PRJNA1009810,Live tracking of basal stem cells of the epidermis during growth  homeostasis  and injury response in zebrafish,GSE241757,Transcriptome Analysis,Basal stem cells of the epidermis continuously differentiate into keratinocytes and replenish themselves via self renewal to maintain skin homeostasis. Numerous studies have attempted to reveal how basal cells undergo differentiation or self renewal; however  this has been hampered by a lack of robust basal cell markers and analytical platforms that allow single cell tracking. Here  we report integrin b4 itgb4 is a useful marker for basal cell labelling  irrespective of the body region  stage  and regenerative status. We employed Cre loxP recombination in combination with live cell tracking of single basal clones in the caudal fin and investigated the embryonic origin and behaviour of basal cells during fish growth and homeostasis. Although most basal cells  including those in fins  became quiescent in the adult stage  genetic cell ablation showed that basal cells were reactivated to either self renew or differentiate  depending on the injured cell type. Our findings provide a platform for quantitative in vivo imaging of basal stem cells applicable at wider stages and under various conditions and reveal unanticipated basal cell dynamism in response to distinct wound signals. Overall design: To investigate whether itgb4+ epidermal basal precursors already possess a basal cell like identity immediately post fate segregation at 1 dpf dpf  we isolated itgb4+ basal precursors  krt4+ periderm cells from zebrafish embryos at 1 dpf  and adult itgb4+ basal cells from zebrafish caudal fin by fluorescence activated cell sorting. We then performed gene expression profiling analysis using data obtained from RNA seq of 3 different cells at 2 time points. Comparative gene expression profiling analysis of RNA seq data for embryonic basal cell precursors  embryonic periderm  and adult basal cells.,,pubmed:38265193,,1 dpf itgb4+ embryonic basal precursors rep1,GSM7734877,,tissue:epidermal cells|cell type:embryonic epidermal basal cells|genotype:Tgitgb4:mcherry|geo loc name:missing|collection date:missing,1 dpf itgb4+ embryonic basal precursors rep1,High quality reads were mapped using hisat2 ver. 2.2.1 Trimmed sequence reads were mapped to GRCz11 using hisat2. Read count extraxction and normalization were performed using featureCounts ver. 2.0.3 Assembly: GRCz11 Supplementary files format and content: tab delimited text file includes basic gene information gene id  length  Genebank id  RNA type  gene name  product  raw count  RPKM and TPM values.,epidermal cells,Different types of cells were sorted by the fluorescence activated cell sorter SH800S SONY.,The sorted cells were directly collected in a lysis buffer and used for RNA extraction using the RNeasy Plus Micro Kit Qiagen RNA libraries were prepared by MGIEasy™ WES rapid library preparation kit according to the manufacturer’s protocol.,Fish were maintained in a recirculating water system in a 14 h day/10 h night photoperiod at 28.5 °C. The wild type zebrafish Danio rerio strain used in this study was originally derived from the Tubingen strain and has been maintained in our facility for more than 10 years by inbreeding.,cell type:embryonic epidermal basal cells|genotype:Tgitgb4:mcherry,GSM7734877,GSM7734877: 1 dpf itgb4+ embryonic basal precursors rep1; Danio rerio; RNA Seq,GSM7734877 r1,GSM7734877,1,The sorted cells were directly collected in a lysis buffer and used for RNA extraction using the RNeasy Plus Micro Kit Qiagen RNA libraries were prepared by MGIEasy™ WES rapid library preparation kit according to the manufacturer's protocol.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,DNBSEQ,DNBSEQ-G400,,SRP457109,,,itgb4mcherry_1_S20_R1_001.fastq.gz itgb4mcherry_1_S20_R2_001.fastq.gz,fastq fastq,7055215600.0,17638039.0,GSM7734877 r1,0:200 1:200,A:1894635584;C:1556854856;G:1769420212;T:1825520802;N:8784146,200,200,,,1894635584,1556854856,1769420212,1825520802,8784146,SRX21486770,SRS18719070,SRA1700438,"Kawakami Lab, School of Life Science and Technology, Tokyo Institute of Technology","Kawakami Lab, School of Life Science and Technology, Tokyo Institute of Technology",2,0.01135,0.01881,0.00043,0.00016,0.9932,0.99543,0.46234,0.43106,200,200,T,T,mates < 9% mapping rate,bgi,bgi,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Japan,2023-08-28,Multi-stage,Multi-stage,Skin,Surface Structure
25300,SRR25764102,SRX21486769,SRS18719069,SRP457109,PRJNA1009810,Live tracking of basal stem cells of the epidermis during growth  homeostasis  and injury response in zebrafish,GSE241757,Transcriptome Analysis,Basal stem cells of the epidermis continuously differentiate into keratinocytes and replenish themselves via self renewal to maintain skin homeostasis. Numerous studies have attempted to reveal how basal cells undergo differentiation or self renewal; however  this has been hampered by a lack of robust basal cell markers and analytical platforms that allow single cell tracking. Here  we report integrin b4 itgb4 is a useful marker for basal cell labelling  irrespective of the body region  stage  and regenerative status. We employed Cre loxP recombination in combination with live cell tracking of single basal clones in the caudal fin and investigated the embryonic origin and behaviour of basal cells during fish growth and homeostasis. Although most basal cells  including those in fins  became quiescent in the adult stage  genetic cell ablation showed that basal cells were reactivated to either self renew or differentiate  depending on the injured cell type. Our findings provide a platform for quantitative in vivo imaging of basal stem cells applicable at wider stages and under various conditions and reveal unanticipated basal cell dynamism in response to distinct wound signals. Overall design: To investigate whether itgb4+ epidermal basal precursors already possess a basal cell like identity immediately post fate segregation at 1 dpf dpf  we isolated itgb4+ basal precursors  krt4+ periderm cells from zebrafish embryos at 1 dpf  and adult itgb4+ basal cells from zebrafish caudal fin by fluorescence activated cell sorting. We then performed gene expression profiling analysis using data obtained from RNA seq of 3 different cells at 2 time points. Comparative gene expression profiling analysis of RNA seq data for embryonic basal cell precursors  embryonic periderm  and adult basal cells.,,pubmed:38265193,,1 dpf krt4+ embryonic periderm cells rep2,GSM7734876,,tissue:epidermal cells|cell type:embryonic periderm cells|genotype:Tgkrt4:creERt2;Olactb:loxP dsRed2 loxP egfp;cryaa:egfp|geo loc name:missing|collection date:missing,1 dpf krt4+ embryonic periderm cells rep2,High quality reads were mapped using hisat2 ver. 2.2.1 Trimmed sequence reads were mapped to GRCz11 using hisat2. Read count extraxction and normalization were performed using featureCounts ver. 2.0.3 Assembly: GRCz11 Supplementary files format and content: tab delimited text file includes basic gene information gene id  length  Genebank id  RNA type  gene name  product  raw count  RPKM and TPM values.,epidermal cells,Different types of cells were sorted by the fluorescence activated cell sorter SH800S SONY.,The sorted cells were directly collected in a lysis buffer and used for RNA extraction using the RNeasy Plus Micro Kit Qiagen RNA libraries were prepared by MGIEasy™ WES rapid library preparation kit according to the manufacturer’s protocol.,Fish were maintained in a recirculating water system in a 14 h day/10 h night photoperiod at 28.5 °C. The wild type zebrafish Danio rerio strain used in this study was originally derived from the Tubingen strain and has been maintained in our facility for more than 10 years by inbreeding.,cell type:embryonic periderm cells|genotype:Tgkrt4:creERt2;Olactb:loxP dsRed2 loxP egfp;cryaa:egfp,GSM7734876,GSM7734876: 1 dpf krt4+ embryonic periderm cells rep2; Danio rerio; RNA Seq,GSM7734876 r1,GSM7734876,1,The sorted cells were directly collected in a lysis buffer and used for RNA extraction using the RNeasy Plus Micro Kit Qiagen RNA libraries were prepared by MGIEasy™ WES rapid library preparation kit according to the manufacturer's protocol.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,DNBSEQ,DNBSEQ-G400,,SRP457109,,,krt4cre-gfp_2_S4_R2_001.fastq.gz krt4cre-gfp_2_S4_R1_001.fastq.gz,fastq fastq,9439207600.0,23598019.0,GSM7734876 r1,0:200 1:200,A:2499982675;C:2086665360;G:2395002073;T:2454282285;N:3275207,200,200,,,2499982675,2086665360,2395002073,2454282285,3275207,SRX21486769,SRS18719069,SRA1700438,"Kawakami Lab, School of Life Science and Technology, Tokyo Institute of Technology","Kawakami Lab, School of Life Science and Technology, Tokyo Institute of Technology",2,0.44931,0.01877,0.02236,0.00115,0.80012,0.99032,0.45473,0.49453,200,200,B,T,mate2 technical by mapping diff,bgi,bgi,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Japan,2023-08-28,Multi-stage,Multi-stage,Skin,Surface Structure
25301,SRR25764103,SRX21486768,SRS18719066,SRP457109,PRJNA1009810,Live tracking of basal stem cells of the epidermis during growth  homeostasis  and injury response in zebrafish,GSE241757,Transcriptome Analysis,Basal stem cells of the epidermis continuously differentiate into keratinocytes and replenish themselves via self renewal to maintain skin homeostasis. Numerous studies have attempted to reveal how basal cells undergo differentiation or self renewal; however  this has been hampered by a lack of robust basal cell markers and analytical platforms that allow single cell tracking. Here  we report integrin b4 itgb4 is a useful marker for basal cell labelling  irrespective of the body region  stage  and regenerative status. We employed Cre loxP recombination in combination with live cell tracking of single basal clones in the caudal fin and investigated the embryonic origin and behaviour of basal cells during fish growth and homeostasis. Although most basal cells  including those in fins  became quiescent in the adult stage  genetic cell ablation showed that basal cells were reactivated to either self renew or differentiate  depending on the injured cell type. Our findings provide a platform for quantitative in vivo imaging of basal stem cells applicable at wider stages and under various conditions and reveal unanticipated basal cell dynamism in response to distinct wound signals. Overall design: To investigate whether itgb4+ epidermal basal precursors already possess a basal cell like identity immediately post fate segregation at 1 dpf dpf  we isolated itgb4+ basal precursors  krt4+ periderm cells from zebrafish embryos at 1 dpf  and adult itgb4+ basal cells from zebrafish caudal fin by fluorescence activated cell sorting. We then performed gene expression profiling analysis using data obtained from RNA seq of 3 different cells at 2 time points. Comparative gene expression profiling analysis of RNA seq data for embryonic basal cell precursors  embryonic periderm  and adult basal cells.,,pubmed:38265193,,1 dpf krt4+ embryonic periderm cells rep1,GSM7734875,,tissue:epidermal cells|cell type:embryonic periderm cells|genotype:Tgkrt4:creERt2;Olactb:loxP dsRed2 loxP egfp;cryaa:egfp|geo loc name:missing|collection date:missing,1 dpf krt4+ embryonic periderm cells rep1,High quality reads were mapped using hisat2 ver. 2.2.1 Trimmed sequence reads were mapped to GRCz11 using hisat2. Read count extraxction and normalization were performed using featureCounts ver. 2.0.3 Assembly: GRCz11 Supplementary files format and content: tab delimited text file includes basic gene information gene id  length  Genebank id  RNA type  gene name  product  raw count  RPKM and TPM values.,epidermal cells,Different types of cells were sorted by the fluorescence activated cell sorter SH800S SONY.,The sorted cells were directly collected in a lysis buffer and used for RNA extraction using the RNeasy Plus Micro Kit Qiagen RNA libraries were prepared by MGIEasy™ WES rapid library preparation kit according to the manufacturer’s protocol.,Fish were maintained in a recirculating water system in a 14 h day/10 h night photoperiod at 28.5 °C. The wild type zebrafish Danio rerio strain used in this study was originally derived from the Tubingen strain and has been maintained in our facility for more than 10 years by inbreeding.,cell type:embryonic periderm cells|genotype:Tgkrt4:creERt2;Olactb:loxP dsRed2 loxP egfp;cryaa:egfp,GSM7734875,GSM7734875: 1 dpf krt4+ embryonic periderm cells rep1; Danio rerio; RNA Seq,GSM7734875 r1,GSM7734875,1,The sorted cells were directly collected in a lysis buffer and used for RNA extraction using the RNeasy Plus Micro Kit Qiagen RNA libraries were prepared by MGIEasy™ WES rapid library preparation kit according to the manufacturer's protocol.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,DNBSEQ,DNBSEQ-G400,,SRP457109,,,krt4cre-gfp_1_S19_R2_001.fastq.gz krt4cre-gfp_1_S19_R1_001.fastq.gz,fastq fastq,6119547200.0,15298868.0,GSM7734875 r1,0:200 1:200,A:1642059769;C:1349462427;G:1535937964;T:1584527048;N:7559992,200,200,,,1642059769,1349462427,1535937964,1584527048,7559992,SRX21486768,SRS18719066,SRA1700438,"Kawakami Lab, School of Life Science and Technology, Tokyo Institute of Technology","Kawakami Lab, School of Life Science and Technology, Tokyo Institute of Technology",2,0.01112,0.01017,0.0003,0.00014,0.9931,0.99586,0.43605,0.40821,200,200,T,T,mates < 9% mapping rate,bgi,bgi,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Japan,2023-08-28,Multi-stage,Multi-stage,Skin,Surface Structure
32858,SRR29482326,SRX24993370,SRS21694834,SRP515140,PRJNA1126247,Specific oncogene activation of the cell of origin in mucosal melanoma [SORT seq],GSE270356,Other,Mucosal melanoma MM is a deadly cancer derived from mucosal melanocytes. To test the consequences of MM genetics  we develop a zebrafish model in which all melanocytes experience CCND1 expression and loss of PTEN and TP53. Surprisingly  melanoma only develops from melanocytes lining internal organs  analogous to the location of patient MM. We find that zebrafish MMs have a unique chromatin landscape from cutaneous melanoma. Internal melanocytes are labeled using a MM specific transcriptional enhancer. Normal zebrafish internal melanocytes share a gene expression signature with MMs. Patient and zebrafish MMs show increased migratory neural crest gene and decreased antigen presentation gene expression  consistent with the increased metastatic behavior and decreased immunotherapy sensitivity of MM. Our work suggests the cell state of the originating melanocyte influences the behavior of derived melanomas. Our animal model phenotypically and transcriptionally mimics patient tumors  allowing this model to be used for MM therapeutic discovery. As this is a non MAPK driven genetically engineered model of melanoma  our work also has implications for the 15% of cutaneous melanoma patients who lack MAPK driving mutations.  Overall design: Single cell RNA sequencing was done on internal vs. external normal adult zebrafish melanocytes using the SORT seq platform.,,,,Internal melanocytes,GSM8340241,,source name:Internal melanocytes|tissue:Internal melanocytes|cell type:melanocytes|genotype:mitfa / ; roy /  fish injected with mitfa:GFP|geo loc name:missing|collection date:missing,Internal melanocytes,BWA was used to align paired end read to danRer11. Count tables were generated using MapAndGo. Count tables were corrected using UMI to remove duplicate reads. Transcript counts were adjusted using Poissonian counting statistics to yield the number of UMIs detected per cell. Counts were imported into R using the Seurat suite version 3.0 Assembly: danRer11 Supplementary files format and content: .tsv file contains count matrix file used for data normalization and visualization using Seurat Library strategy: SORT seq,Internal melanocytes,,Respective tissues were mechanically dissociated  digested in TrypLE for 45 min Invitrogen  12563011  40uM filtered  spun down  and resuspended in FACs buffer dPBS Mg+/Ca+ free  with 2% FBS  pen/strep. Cells were heat lysed at 65°C followed by cDNA synthesis with barcodes. All the barcoded material from one plate was pooled into one library and amplified using in vitro transcription. Following amplification  library preparation was done following the CEL Seq2 protocol to prepare a cDNA library for sequencing using TruSeq small RNA primers Illumina.,mitfa / ; roy /  zebrafish injected with mitfa:GFP were Raised to maturity to obtain tissues,tissue:Internal melanocytes|cell type:melanocytes|genotype:mitfa / ; roy /  fish injected with mitfa:GFP,GSM8340241,GSM8340241: Internal melanocytes; Danio rerio; OTHER,GSM8340241 r1,GSM8340241,1,Respective tissues were mechanically dissociated  digested in TrypLE for 45 min Invitrogen  12563011  40uM filtered  spun down  and resuspended in FACs buffer dPBS Mg+/Ca+ free  with 2% FBS  pen/strep. Cells were heat lysed at 65°C followed by cDNA synthesis with barcodes. All the barcoded material from one plate was pooled into one library and amplified using in vitro transcription. Following amplification  library preparation was done following the CEL Seq2 protocol to prepare a cDNA library for sequencing using TruSeq small RNA primers Illumina.,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,NextSeq 500,,SRP515140,,,HAR-MI-002_H5GCWBGXF_R2.fastq.gz HAR-MI-002_H5GCWBGXF_R1.fastq.gz,fastq fastq,2957975418.0,34395063.0,GSM8340241 r1,0:26 1:60,A:738954503;C:560052455;G:520746060;T:1137116132;N:1106268,26,60,,,738954503,560052455,520746060,1137116132,1106268,SRX24993370,SRS21694834,SRA1904773,"Insco Lab, Medical Oncology, Dana Farber Cancer Institute","Insco Lab, Medical Oncology, Dana Farber Cancer Institute",2,0.11637,0.84116,0.10894,0.31188,0.98851,0.71526,0.6688,0.60221,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,United States,2024-06-20,Undetermined,Adult,Skin,Surface Structure
32859,SRR29482327,SRX24993369,SRS21694833,SRP515140,PRJNA1126247,Specific oncogene activation of the cell of origin in mucosal melanoma [SORT seq],GSE270356,Other,Mucosal melanoma MM is a deadly cancer derived from mucosal melanocytes. To test the consequences of MM genetics  we develop a zebrafish model in which all melanocytes experience CCND1 expression and loss of PTEN and TP53. Surprisingly  melanoma only develops from melanocytes lining internal organs  analogous to the location of patient MM. We find that zebrafish MMs have a unique chromatin landscape from cutaneous melanoma. Internal melanocytes are labeled using a MM specific transcriptional enhancer. Normal zebrafish internal melanocytes share a gene expression signature with MMs. Patient and zebrafish MMs show increased migratory neural crest gene and decreased antigen presentation gene expression  consistent with the increased metastatic behavior and decreased immunotherapy sensitivity of MM. Our work suggests the cell state of the originating melanocyte influences the behavior of derived melanomas. Our animal model phenotypically and transcriptionally mimics patient tumors  allowing this model to be used for MM therapeutic discovery. As this is a non MAPK driven genetically engineered model of melanoma  our work also has implications for the 15% of cutaneous melanoma patients who lack MAPK driving mutations.  Overall design: Single cell RNA sequencing was done on internal vs. external normal adult zebrafish melanocytes using the SORT seq platform.,,,,Cutaneous melanocytes,GSM8340240,,source name:Cutaneous melanocytes|tissue:Cutaneous melanocytes|cell type:melanocytes|genotype:mitfa / ; roy /  fish injected with mitfa:GFP|geo loc name:missing|collection date:missing,Cutaneous melanocytes,BWA was used to align paired end read to danRer11. Count tables were generated using MapAndGo. Count tables were corrected using UMI to remove duplicate reads. Transcript counts were adjusted using Poissonian counting statistics to yield the number of UMIs detected per cell. Counts were imported into R using the Seurat suite version 3.0 Assembly: danRer11 Supplementary files format and content: .tsv file contains count matrix file used for data normalization and visualization using Seurat Library strategy: SORT seq,Cutaneous melanocytes,,Respective tissues were mechanically dissociated  digested in TrypLE for 45 min Invitrogen  12563011  40uM filtered  spun down  and resuspended in FACs buffer dPBS Mg+/Ca+ free  with 2% FBS  pen/strep. Cells were heat lysed at 65°C followed by cDNA synthesis with barcodes. All the barcoded material from one plate was pooled into one library and amplified using in vitro transcription. Following amplification  library preparation was done following the CEL Seq2 protocol to prepare a cDNA library for sequencing using TruSeq small RNA primers Illumina.,mitfa / ; roy /  zebrafish injected with mitfa:GFP were Raised to maturity to obtain tissues,tissue:Cutaneous melanocytes|cell type:melanocytes|genotype:mitfa / ; roy /  fish injected with mitfa:GFP,GSM8340240,GSM8340240: Cutaneous melanocytes; Danio rerio; OTHER,GSM8340240 r1,GSM8340240,1,Respective tissues were mechanically dissociated  digested in TrypLE for 45 min Invitrogen  12563011  40uM filtered  spun down  and resuspended in FACs buffer dPBS Mg+/Ca+ free  with 2% FBS  pen/strep. Cells were heat lysed at 65°C followed by cDNA synthesis with barcodes. All the barcoded material from one plate was pooled into one library and amplified using in vitro transcription. Following amplification  library preparation was done following the CEL Seq2 protocol to prepare a cDNA library for sequencing using TruSeq small RNA primers Illumina.,,OTHER,TRANSCRIPTOMIC,other,PAIRED,ILLUMINA,NextSeq 500,,SRP515140,,,HAR-MI-001_H5GCWBGXF_R2.fastq.gz HAR-MI-001_H5GCWBGXF_R1.fastq.gz,fastq fastq,2680460212.0,31168142.0,GSM8340240 r1,0:26 1:60,A:704357434;C:519158361;G:467098420;T:988838604;N:1007393,26,60,,,704357434,519158361,467098420,988838604,1007393,SRX24993369,SRS21694833,SRA1904773,"Insco Lab, Medical Oncology, Dana Farber Cancer Institute","Insco Lab, Medical Oncology, Dana Farber Cancer Institute",2,0.11679,0.81545,0.10693,0.5033,0.9808,0.76404,0.44749,0.57623,26,60,T,B,sc-like readlen,illumina,nextseq,unknown,small_rna,trueseq,sc,single_cell_plate,celseq,,United States,2024-06-20,Undetermined,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&amp;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&amp;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&amp;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&amp;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&amp;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&amp;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&amp;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&amp;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&amp;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
33894,SRR30866028,SRX26263941,SRS22803195,SRP536275,PRJNA1168148,Mgat4b mediated selective N glycosyl modification regulates melanocyte development and melanoma progression [bulk RNA seq],GSE278653,Transcriptome Analysis,Dysregulated melanocyte state transitions are a pivotal driver of melanoma development  highlighting the need to identify key regulators of these processes. Understanding these factors is key to know how normal melanocyte functions and shift towards initiation of melanoma. Our study identifies Mgat4b  a glycosyl transferase involved in selective N glycan branching enriched in pigment progenitors  as a key regulator of directional melanocyte migration and establishment of Melanocyte stem cell McSC pool during early development in zebrafish and mammalian melanocytes. Single cell RNA sequencing analysis in zebrafish upon targeted disruption of Mgat4b reveals that a subset of melanocytes marked by aberrant galectin expression are impaired in migration and are lost. Lectin binding proteomic analysis reveals the glycosylation of key melanocyte proteins Gpnmb  Kit  and Tyrp1 to be under the control of Mgat4b. Additionally  mislocalization of Gamma catenin Jup explains the observed defects in cell adhesion and migration to be regulated by mgat4b but not its isozyme mgat4a. Our meta analysis further revealed that melanoma patients with both the BrafV600E mutation and elevated Mgat4b levels have significantly worse survival outcomes compared to those with only the BrafV600E mutation. By leveraging the MAZERATI platform to model BrafV600E driver mutation in vivo  we show that Mgat4b mutant cells fail to aggregate and initiate tumors. Our study underscores the importance of selective N glycan branching in both melanocyte development and melanoma initiation  suggesting a Mitf controlled Mgat4b as a promising therapeutic target for melanoma treatment. Overall design: To investigate the mechanisms that inhibit mutant cells from initiating melanoma in the absence of mgat4b  we utilized mature melanophores and melanoma cells from the skin of five zebrafish  including both MAZERATI wild type and  MAZERATI zebrafish carrying mgat4b mutations. We then performed gene expression profiling analysis using data obtained from RNA seq of melanophores and melanoma cells from both the zebrafish wild type and m4b mut. Comparative gene expression analysis was performed for wild type melanoma and melanophores  m4b mut melanoma and melanophores  wild type and m4b mut melanoma.,,,,m4b mut melanoma  biol rep 2,GSM8552314,,source name:skin|tissue:skin|cell type:melanoma|genotype:mgat4b mutant|treatment:MAZERATI|geo loc name:missing|collection date:missing,m4b mut melanoma  biol rep 2,Raw fastq files were trimmed using trimmomatic to remove adaptors and retain high quality reads. The reads were then aligned with zebrafish reference assembly GRCz11 using STAR. Featurecounts was used to calculate raw counts from the aligned reads. Assembly: GRCz11 Supplementary files format and content: tab delimited text file including raw counts for each sample,skin,,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,tissue:skin|cell type:melanoma|genotype:mgat4b mutant|treatment:MAZERATI,GSM8552314,GSM8552314: m4b mut melanoma  biol rep 2; Danio rerio; RNA Seq,GSM8552314 r1,GSM8552314,1,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 2000,,SRP536275,,,MKO2_S39_R1_001.fastq.gz MKO2_S39_R2_001.fastq.gz,fastq fastq,3705364625.0,14164066.0,GSM8552314 r1,0:126.72 1:134.89,A:785806700;C:949078797;G:1178279032;T:784972987;N:7227109,126,134,,,785806700,949078797,1178279032,784972987,7227109,SRX26263941,SRS22803195,SRA1984938,"Pigment Cell Biology Lab, CSIR-IGIB","Pigment Cell Biology Lab, CSIR-IGIB",,,,,,,,,,,,B,B,biological fallback assumption,illumina,nextseq_v2,unknown,cdna_unspecified,trueseq,sc_generic,bulk,bulk,,India,2024-10-02,Undetermined,Undetermined,Skin,Surface Structure
33895,SRR30866029,SRX26263940,SRS22803194,SRP536275,PRJNA1168148,Mgat4b mediated selective N glycosyl modification regulates melanocyte development and melanoma progression [bulk RNA seq],GSE278653,Transcriptome Analysis,Dysregulated melanocyte state transitions are a pivotal driver of melanoma development  highlighting the need to identify key regulators of these processes. Understanding these factors is key to know how normal melanocyte functions and shift towards initiation of melanoma. Our study identifies Mgat4b  a glycosyl transferase involved in selective N glycan branching enriched in pigment progenitors  as a key regulator of directional melanocyte migration and establishment of Melanocyte stem cell McSC pool during early development in zebrafish and mammalian melanocytes. Single cell RNA sequencing analysis in zebrafish upon targeted disruption of Mgat4b reveals that a subset of melanocytes marked by aberrant galectin expression are impaired in migration and are lost. Lectin binding proteomic analysis reveals the glycosylation of key melanocyte proteins Gpnmb  Kit  and Tyrp1 to be under the control of Mgat4b. Additionally  mislocalization of Gamma catenin Jup explains the observed defects in cell adhesion and migration to be regulated by mgat4b but not its isozyme mgat4a. Our meta analysis further revealed that melanoma patients with both the BrafV600E mutation and elevated Mgat4b levels have significantly worse survival outcomes compared to those with only the BrafV600E mutation. By leveraging the MAZERATI platform to model BrafV600E driver mutation in vivo  we show that Mgat4b mutant cells fail to aggregate and initiate tumors. Our study underscores the importance of selective N glycan branching in both melanocyte development and melanoma initiation  suggesting a Mitf controlled Mgat4b as a promising therapeutic target for melanoma treatment. Overall design: To investigate the mechanisms that inhibit mutant cells from initiating melanoma in the absence of mgat4b  we utilized mature melanophores and melanoma cells from the skin of five zebrafish  including both MAZERATI wild type and  MAZERATI zebrafish carrying mgat4b mutations. We then performed gene expression profiling analysis using data obtained from RNA seq of melanophores and melanoma cells from both the zebrafish wild type and m4b mut. Comparative gene expression analysis was performed for wild type melanoma and melanophores  m4b mut melanoma and melanophores  wild type and m4b mut melanoma.,,,,m4b mut melanoma  biol rep 1,GSM8552313,,source name:skin|tissue:skin|cell type:melanoma|genotype:mgat4b mutant|treatment:MAZERATI|geo loc name:missing|collection date:missing,m4b mut melanoma  biol rep 1,Raw fastq files were trimmed using trimmomatic to remove adaptors and retain high quality reads. The reads were then aligned with zebrafish reference assembly GRCz11 using STAR. Featurecounts was used to calculate raw counts from the aligned reads. Assembly: GRCz11 Supplementary files format and content: tab delimited text file including raw counts for each sample,skin,,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,tissue:skin|cell type:melanoma|genotype:mgat4b mutant|treatment:MAZERATI,GSM8552313,GSM8552313: m4b mut melanoma  biol rep 1; Danio rerio; RNA Seq,GSM8552313 r1,GSM8552313,1,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 2000,,SRP536275,,,MKO1_S38_R1_001.fastq.gz MKO1_S38_R2_001.fastq.gz,fastq fastq,4779377372.0,18058294.0,GSM8552313 r1,0:128.72 1:135.94,A:1016908920;C:1236613888;G:1502465334;T:1018053808;N:5335422,128,135,,,1016908920,1236613888,1502465334,1018053808,5335422,SRX26263940,SRS22803194,SRA1984938,"Pigment Cell Biology Lab, CSIR-IGIB","Pigment Cell Biology Lab, CSIR-IGIB",,,,,,,,,,,,B,B,biological fallback assumption,illumina,nextseq_v2,unknown,cdna_unspecified,trueseq,sc_generic,bulk,bulk,,India,2024-10-02,Undetermined,Undetermined,Skin,Surface Structure
33896,SRR30866030,SRX26263939,SRS22803193,SRP536275,PRJNA1168148,Mgat4b mediated selective N glycosyl modification regulates melanocyte development and melanoma progression [bulk RNA seq],GSE278653,Transcriptome Analysis,Dysregulated melanocyte state transitions are a pivotal driver of melanoma development  highlighting the need to identify key regulators of these processes. Understanding these factors is key to know how normal melanocyte functions and shift towards initiation of melanoma. Our study identifies Mgat4b  a glycosyl transferase involved in selective N glycan branching enriched in pigment progenitors  as a key regulator of directional melanocyte migration and establishment of Melanocyte stem cell McSC pool during early development in zebrafish and mammalian melanocytes. Single cell RNA sequencing analysis in zebrafish upon targeted disruption of Mgat4b reveals that a subset of melanocytes marked by aberrant galectin expression are impaired in migration and are lost. Lectin binding proteomic analysis reveals the glycosylation of key melanocyte proteins Gpnmb  Kit  and Tyrp1 to be under the control of Mgat4b. Additionally  mislocalization of Gamma catenin Jup explains the observed defects in cell adhesion and migration to be regulated by mgat4b but not its isozyme mgat4a. Our meta analysis further revealed that melanoma patients with both the BrafV600E mutation and elevated Mgat4b levels have significantly worse survival outcomes compared to those with only the BrafV600E mutation. By leveraging the MAZERATI platform to model BrafV600E driver mutation in vivo  we show that Mgat4b mutant cells fail to aggregate and initiate tumors. Our study underscores the importance of selective N glycan branching in both melanocyte development and melanoma initiation  suggesting a Mitf controlled Mgat4b as a promising therapeutic target for melanoma treatment. Overall design: To investigate the mechanisms that inhibit mutant cells from initiating melanoma in the absence of mgat4b  we utilized mature melanophores and melanoma cells from the skin of five zebrafish  including both MAZERATI wild type and  MAZERATI zebrafish carrying mgat4b mutations. We then performed gene expression profiling analysis using data obtained from RNA seq of melanophores and melanoma cells from both the zebrafish wild type and m4b mut. Comparative gene expression analysis was performed for wild type melanoma and melanophores  m4b mut melanoma and melanophores  wild type and m4b mut melanoma.,,,,wild type melanoma control  biol rep 2,GSM8552312,,source name:skin|tissue:skin|cell type:melanoma|genotype:WT|treatment:MAZERATI|geo loc name:missing|collection date:missing,wild type melanoma control  biol rep 2,Raw fastq files were trimmed using trimmomatic to remove adaptors and retain high quality reads. The reads were then aligned with zebrafish reference assembly GRCz11 using STAR. Featurecounts was used to calculate raw counts from the aligned reads. Assembly: GRCz11 Supplementary files format and content: tab delimited text file including raw counts for each sample,skin,,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,tissue:skin|cell type:melanoma|genotype:WT|treatment:MAZERATI,GSM8552312,GSM8552312: wild type melanoma control  biol rep 2; Danio rerio; RNA Seq,GSM8552312 r1,GSM8552312,1,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 2000,,SRP536275,,,EV2_S37_R1_001.fastq.gz EV2_S37_R2_001.fastq.gz,fastq fastq,5427379509.0,19981504.0,GSM8552312 r1,0:133.16 1:138.46,A:1136639418;C:1414638483;G:1736492639;T:1137614638;N:1994331,133,138,,,1136639418,1414638483,1736492639,1137614638,1994331,SRX26263939,SRS22803193,SRA1984938,"Pigment Cell Biology Lab, CSIR-IGIB","Pigment Cell Biology Lab, CSIR-IGIB",,,,,,,,,,,,B,B,biological fallback assumption,illumina,nextseq_v2,unknown,cdna_unspecified,trueseq,sc_generic,bulk,bulk,,India,2024-10-02,Undetermined,Undetermined,Skin,Surface Structure
33897,SRR30866031,SRX26263938,SRS22803192,SRP536275,PRJNA1168148,Mgat4b mediated selective N glycosyl modification regulates melanocyte development and melanoma progression [bulk RNA seq],GSE278653,Transcriptome Analysis,Dysregulated melanocyte state transitions are a pivotal driver of melanoma development  highlighting the need to identify key regulators of these processes. Understanding these factors is key to know how normal melanocyte functions and shift towards initiation of melanoma. Our study identifies Mgat4b  a glycosyl transferase involved in selective N glycan branching enriched in pigment progenitors  as a key regulator of directional melanocyte migration and establishment of Melanocyte stem cell McSC pool during early development in zebrafish and mammalian melanocytes. Single cell RNA sequencing analysis in zebrafish upon targeted disruption of Mgat4b reveals that a subset of melanocytes marked by aberrant galectin expression are impaired in migration and are lost. Lectin binding proteomic analysis reveals the glycosylation of key melanocyte proteins Gpnmb  Kit  and Tyrp1 to be under the control of Mgat4b. Additionally  mislocalization of Gamma catenin Jup explains the observed defects in cell adhesion and migration to be regulated by mgat4b but not its isozyme mgat4a. Our meta analysis further revealed that melanoma patients with both the BrafV600E mutation and elevated Mgat4b levels have significantly worse survival outcomes compared to those with only the BrafV600E mutation. By leveraging the MAZERATI platform to model BrafV600E driver mutation in vivo  we show that Mgat4b mutant cells fail to aggregate and initiate tumors. Our study underscores the importance of selective N glycan branching in both melanocyte development and melanoma initiation  suggesting a Mitf controlled Mgat4b as a promising therapeutic target for melanoma treatment. Overall design: To investigate the mechanisms that inhibit mutant cells from initiating melanoma in the absence of mgat4b  we utilized mature melanophores and melanoma cells from the skin of five zebrafish  including both MAZERATI wild type and  MAZERATI zebrafish carrying mgat4b mutations. We then performed gene expression profiling analysis using data obtained from RNA seq of melanophores and melanoma cells from both the zebrafish wild type and m4b mut. Comparative gene expression analysis was performed for wild type melanoma and melanophores  m4b mut melanoma and melanophores  wild type and m4b mut melanoma.,,,,wild type melanoma control  biol rep 1,GSM8552311,,source name:skin|tissue:skin|cell type:melanoma|genotype:WT|treatment:MAZERATI|geo loc name:missing|collection date:missing,wild type melanoma control  biol rep 1,Raw fastq files were trimmed using trimmomatic to remove adaptors and retain high quality reads. The reads were then aligned with zebrafish reference assembly GRCz11 using STAR. Featurecounts was used to calculate raw counts from the aligned reads. Assembly: GRCz11 Supplementary files format and content: tab delimited text file including raw counts for each sample,skin,,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,tissue:skin|cell type:melanoma|genotype:WT|treatment:MAZERATI,GSM8552311,GSM8552311: wild type melanoma control  biol rep 1; Danio rerio; RNA Seq,GSM8552311 r1,GSM8552311,1,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 2000,,SRP536275,,,EV1_S36_R1_001.fastq.gz EV1_S36_R2_001.fastq.gz,fastq fastq,5248108957.0,19087024.0,GSM8552311 r1,0:135.12 1:139.84,A:1112163842;C:1354895660;G:1658732932;T:1120342985;N:1973538,135,139,,,1112163842,1354895660,1658732932,1120342985,1973538,SRX26263938,SRS22803192,SRA1984938,"Pigment Cell Biology Lab, CSIR-IGIB","Pigment Cell Biology Lab, CSIR-IGIB",,,,,,,,,,,,B,B,biological fallback assumption,illumina,nextseq_v2,unknown,cdna_unspecified,trueseq,sc_generic,bulk,bulk,,India,2024-10-02,Undetermined,Undetermined,Skin,Surface Structure
33898,SRR30866032,SRX26263937,SRS22803191,SRP536275,PRJNA1168148,Mgat4b mediated selective N glycosyl modification regulates melanocyte development and melanoma progression [bulk RNA seq],GSE278653,Transcriptome Analysis,Dysregulated melanocyte state transitions are a pivotal driver of melanoma development  highlighting the need to identify key regulators of these processes. Understanding these factors is key to know how normal melanocyte functions and shift towards initiation of melanoma. Our study identifies Mgat4b  a glycosyl transferase involved in selective N glycan branching enriched in pigment progenitors  as a key regulator of directional melanocyte migration and establishment of Melanocyte stem cell McSC pool during early development in zebrafish and mammalian melanocytes. Single cell RNA sequencing analysis in zebrafish upon targeted disruption of Mgat4b reveals that a subset of melanocytes marked by aberrant galectin expression are impaired in migration and are lost. Lectin binding proteomic analysis reveals the glycosylation of key melanocyte proteins Gpnmb  Kit  and Tyrp1 to be under the control of Mgat4b. Additionally  mislocalization of Gamma catenin Jup explains the observed defects in cell adhesion and migration to be regulated by mgat4b but not its isozyme mgat4a. Our meta analysis further revealed that melanoma patients with both the BrafV600E mutation and elevated Mgat4b levels have significantly worse survival outcomes compared to those with only the BrafV600E mutation. By leveraging the MAZERATI platform to model BrafV600E driver mutation in vivo  we show that Mgat4b mutant cells fail to aggregate and initiate tumors. Our study underscores the importance of selective N glycan branching in both melanocyte development and melanoma initiation  suggesting a Mitf controlled Mgat4b as a promising therapeutic target for melanoma treatment. Overall design: To investigate the mechanisms that inhibit mutant cells from initiating melanoma in the absence of mgat4b  we utilized mature melanophores and melanoma cells from the skin of five zebrafish  including both MAZERATI wild type and  MAZERATI zebrafish carrying mgat4b mutations. We then performed gene expression profiling analysis using data obtained from RNA seq of melanophores and melanoma cells from both the zebrafish wild type and m4b mut. Comparative gene expression analysis was performed for wild type melanoma and melanophores  m4b mut melanoma and melanophores  wild type and m4b mut melanoma.,,,,melanophore  biol rep 2,GSM8552310,,source name:skin|tissue:skin|cell type:melanophore|genotype:WT|treatment:No|geo loc name:missing|collection date:missing,melanophore  biol rep 2,Raw fastq files were trimmed using trimmomatic to remove adaptors and retain high quality reads. The reads were then aligned with zebrafish reference assembly GRCz11 using STAR. Featurecounts was used to calculate raw counts from the aligned reads. Assembly: GRCz11 Supplementary files format and content: tab delimited text file including raw counts for each sample,skin,,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,tissue:skin|cell type:melanophore|genotype:WT|treatment:No,GSM8552310,GSM8552310: melanophore  biol rep 2; Danio rerio; RNA Seq,GSM8552310 r1,GSM8552310,1,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 2000,,SRP536275,,,WT2_S41_R1_001.fastq.gz WT2_S41_R2_001.fastq.gz,fastq fastq,4636631881.0,16340506.0,GSM8552310 r1,0:140.47 1:143.28,A:1114533149;C:1104066636;G:1293649625;T:1122672029;N:1710442,140,143,,,1114533149,1104066636,1293649625,1122672029,1710442,SRX26263937,SRS22803191,SRA1984938,"Pigment Cell Biology Lab, CSIR-IGIB","Pigment Cell Biology Lab, CSIR-IGIB",,,,,,,,,,,,B,B,biological fallback assumption,illumina,nextseq_v2,unknown,cdna_unspecified,trueseq,sc_generic,bulk,bulk,,India,2024-10-02,Undetermined,Undetermined,Skin,Surface Structure
33899,SRR30866033,SRX26263936,SRS22803190,SRP536275,PRJNA1168148,Mgat4b mediated selective N glycosyl modification regulates melanocyte development and melanoma progression [bulk RNA seq],GSE278653,Transcriptome Analysis,Dysregulated melanocyte state transitions are a pivotal driver of melanoma development  highlighting the need to identify key regulators of these processes. Understanding these factors is key to know how normal melanocyte functions and shift towards initiation of melanoma. Our study identifies Mgat4b  a glycosyl transferase involved in selective N glycan branching enriched in pigment progenitors  as a key regulator of directional melanocyte migration and establishment of Melanocyte stem cell McSC pool during early development in zebrafish and mammalian melanocytes. Single cell RNA sequencing analysis in zebrafish upon targeted disruption of Mgat4b reveals that a subset of melanocytes marked by aberrant galectin expression are impaired in migration and are lost. Lectin binding proteomic analysis reveals the glycosylation of key melanocyte proteins Gpnmb  Kit  and Tyrp1 to be under the control of Mgat4b. Additionally  mislocalization of Gamma catenin Jup explains the observed defects in cell adhesion and migration to be regulated by mgat4b but not its isozyme mgat4a. Our meta analysis further revealed that melanoma patients with both the BrafV600E mutation and elevated Mgat4b levels have significantly worse survival outcomes compared to those with only the BrafV600E mutation. By leveraging the MAZERATI platform to model BrafV600E driver mutation in vivo  we show that Mgat4b mutant cells fail to aggregate and initiate tumors. Our study underscores the importance of selective N glycan branching in both melanocyte development and melanoma initiation  suggesting a Mitf controlled Mgat4b as a promising therapeutic target for melanoma treatment. Overall design: To investigate the mechanisms that inhibit mutant cells from initiating melanoma in the absence of mgat4b  we utilized mature melanophores and melanoma cells from the skin of five zebrafish  including both MAZERATI wild type and  MAZERATI zebrafish carrying mgat4b mutations. We then performed gene expression profiling analysis using data obtained from RNA seq of melanophores and melanoma cells from both the zebrafish wild type and m4b mut. Comparative gene expression analysis was performed for wild type melanoma and melanophores  m4b mut melanoma and melanophores  wild type and m4b mut melanoma.,,,,melanophore  biol rep 1,GSM8552309,,source name:skin|tissue:skin|cell type:melanophore|genotype:WT|treatment:No|geo loc name:missing|collection date:missing,melanophore  biol rep 1,Raw fastq files were trimmed using trimmomatic to remove adaptors and retain high quality reads. The reads were then aligned with zebrafish reference assembly GRCz11 using STAR. Featurecounts was used to calculate raw counts from the aligned reads. Assembly: GRCz11 Supplementary files format and content: tab delimited text file including raw counts for each sample,skin,,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,tissue:skin|cell type:melanophore|genotype:WT|treatment:No,GSM8552309,GSM8552309: melanophore  biol rep 1; Danio rerio; RNA Seq,GSM8552309 r1,GSM8552309,1,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 2000,,SRP536275,,,WT1_S40_R1_001.fastq.gz WT1_S40_R2_001.fastq.gz,fastq fastq,5185761246.0,18118534.0,GSM8552309 r1,0:141.81 1:144.40,A:1231235669;C:1235759770;G:1476864583;T:1240636871;N:1264353,141,144,,,1231235669,1235759770,1476864583,1240636871,1264353,SRX26263936,SRS22803190,SRA1984938,"Pigment Cell Biology Lab, CSIR-IGIB","Pigment Cell Biology Lab, CSIR-IGIB",,,,,,,,,,,,B,B,biological fallback assumption,illumina,nextseq_v2,unknown,cdna_unspecified,trueseq,sc_generic,bulk,bulk,,India,2024-10-02,Undetermined,Undetermined,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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
36741,SRR850608,SRX277695,SRS420758,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr18 danrer skin 42months rep5,GSM1141118,,source name:total RNA extracted from skin|strain:TuAB|age:42 month|tissue:skin,Nr18 danrer skin 42months rep5,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:42 month|tissue:skin,GSM1141118,GSM1141118: Nr18 danrer skin 42months rep5; Danio rerio; RNA Seq,GSM1141118 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141118,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr18_3.5y_l3_export.fq.gz,fastq,3269418540.0,43018665.0,GSM1141118 r1,0:76,A:892486836;C:744426899;G:749230539;T:883114979;N:159287,76,,,,892486836,744426899,749230539,883114979,159287,SRX277695,SRS420758,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91402,,0.1106,,0.73436,,0.4911,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36742,SRR850607,SRX277694,SRS420757,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr14 danrer skin 42months rep4,GSM1141117,,source name:total RNA extracted from skin|strain:TuAB|age:42 month|tissue:skin,Nr14 danrer skin 42months rep4,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:42 month|tissue:skin,GSM1141117,GSM1141117: Nr14 danrer skin 42months rep4; Danio rerio; RNA Seq,GSM1141117 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141117,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr14_3.5y_l2_export.fq.gz,fastq,2300890044.0,30274869.0,GSM1141117 r1,0:76,A:631736236;C:525065199;G:510761115;T:633208038;N:119456,76,,,,631736236,525065199,510761115,633208038,119456,SRX277694,SRS420757,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.90718,,0.11607,,0.71997,,0.49141,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36743,SRR850606,SRX277693,SRS420790,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr13 danrer skin 42months rep3,GSM1141116,,source name:total RNA extracted from skin|strain:TuAB|age:42 month|tissue:skin,Nr13 danrer skin 42months rep3,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:42 month|tissue:skin,GSM1141116,GSM1141116: Nr13 danrer skin 42months rep3; Danio rerio; RNA Seq,GSM1141116 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141116,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr13_3.5y_l1_export.fastq.gz,fastq,3508110372.0,46159347.0,GSM1141116 r1,0:76,A:959986155;C:782258637;G:803256847;T:962450623;N:158110,76,,,,959986155,782258637,803256847,962450623,158110,SRX277693,SRS420790,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.85747,,0.12068,,0.73819,,0.4917,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36744,SRR850605,SRX277692,SRS420789,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr12 danrer skin 42months rep2,GSM1141115,,source name:total RNA extracted from skin|strain:TuAB|age:42 month|tissue:skin,Nr12 danrer skin 42months rep2,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:42 month|tissue:skin,GSM1141115,GSM1141115: Nr12 danrer skin 42months rep2; Danio rerio; RNA Seq,GSM1141115 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141115,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr12_3.5y_l3_export.fq.gz,fastq,3961243804.0,52121629.0,GSM1141115 r1,0:76,A:1092811778;C:874440215;G:892629531;T:1099367104;N:1995176,76,,,,1092811778,874440215,892629531,1099367104,1995176,SRX277692,SRS420789,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.88854,,0.12633,,0.73746,,0.53849,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36745,SRR850604,SRX277691,SRS420788,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr11 danrer skin 42months rep1,GSM1141114,,source name:total RNA extracted from skin|strain:TuAB|age:42 month|tissue:skin,Nr11 danrer skin 42months rep1,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:42 month|tissue:skin,GSM1141114,GSM1141114: Nr11 danrer skin 42months rep1; Danio rerio; RNA Seq,GSM1141114 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141114,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr11_3.5y_l2_export.fq.gz,fastq,3327777040.0,43786540.0,GSM1141114 r1,0:76,A:920364748;C:745739060;G:758842746;T:901558784;N:1271702,76,,,,920364748,745739060,758842746,901558784,1271702,SRX277691,SRS420788,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.86781,,0.11618,,0.72888,,0.50217,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36746,SRR850603,SRX277690,SRS420756,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr76 danrer skin 24months rep6,GSM1141113,,source name:total RNA extracted from skin|strain:TuAB|age:24 month|tissue:skin,Nr76 danrer skin 24months rep6,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:24 month|tissue:skin,GSM1141113,GSM1141113: Nr76 danrer skin 24months rep6; Danio rerio; RNA Seq,GSM1141113 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141113,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr76_2y.fastq.gz,fastq,2234765940.0,29404815.0,GSM1141113 r1,0:76,A:582794172;C:524083367;G:547601836;T:579473821;N:812744,76,,,,582794172,524083367,547601836,579473821,812744,SRX277690,SRS420756,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.89808,,0.07989,,0.73511,,0.47456,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36747,SRR850602,SRX277689,SRS420755,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr75 danrer skin 24months rep5,GSM1141112,,source name:total RNA extracted from skin|strain:TuAB|age:24 month|tissue:skin,Nr75 danrer skin 24months rep5,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:24 month|tissue:skin,GSM1141112,GSM1141112: Nr75 danrer skin 24months rep5; Danio rerio; RNA Seq,GSM1141112 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141112,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr75_2y.fastq.gz,fastq,1000606196.0,13165871.0,GSM1141112 r1,0:76,A:270569436;C:233353660;G:238144829;T:258045669;N:492602,76,,,,270569436,233353660,238144829,258045669,492602,SRX277689,SRS420755,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.8949,,0.07966,,0.7293,,0.47729,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36748,SRR850601,SRX277688,SRS420754,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr73 danrer skin 24months rep4,GSM1141111,,source name:total RNA extracted from skin|strain:TuAB|age:24 month|tissue:skin,Nr73 danrer skin 24months rep4,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:24 month|tissue:skin,GSM1141111,GSM1141111: Nr73 danrer skin 24months rep4; Danio rerio; RNA Seq,GSM1141111 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141111,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr73_2y.fastq.gz,fastq,2283103688.0,30040838.0,GSM1141111 r1,0:76,A:601138588;C:527906584;G:549863593;T:603391353;N:803570,76,,,,601138588,527906584,549863593,603391353,803570,SRX277688,SRS420754,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.90138,,0.08154,,0.72598,,0.49455,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36749,SRR850600,SRX277687,SRS420787,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr72 danrer skin 24months rep3,GSM1141110,,source name:total RNA extracted from skin|strain:TuAB|age:24 month|tissue:skin,Nr72 danrer skin 24months rep3,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:24 month|tissue:skin,GSM1141110,GSM1141110: Nr72 danrer skin 24months rep3; Danio rerio; RNA Seq,GSM1141110 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141110,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr72_2y.fastq.gz,fastq,1692545916.0,22270341.0,GSM1141110 r1,0:76,A:447927627;C:393400289;G:405212681;T:445305247;N:700072,76,,,,447927627,393400289,405212681,445305247,700072,SRX277687,SRS420787,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.88704,,0.08604,,0.72833,,0.47215,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36750,SRR850599,SRX277686,SRS420786,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr71 danrer skin 24months rep2,GSM1141109,,source name:total RNA extracted from skin|strain:TuAB|age:24 month|tissue:skin,Nr71 danrer skin 24months rep2,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:24 month|tissue:skin,GSM1141109,GSM1141109: Nr71 danrer skin 24months rep2; Danio rerio; RNA Seq,GSM1141109 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141109,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr71_2y.fastq.gz,fastq,1897129404.0,24962229.0,GSM1141109 r1,0:76,A:500740954;C:442627409;G:457159802;T:495689680;N:911559,76,,,,500740954,442627409,457159802,495689680,911559,SRX277686,SRS420786,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.88731,,0.07537,,0.73628,,0.4846,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36751,SRR850598,SRX277685,SRS420785,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr70 danrer skin 24months rep1,GSM1141108,,source name:total RNA extracted from skin|strain:TuAB|age:24 month|tissue:skin,Nr70 danrer skin 24months rep1,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:24 month|tissue:skin,GSM1141108,GSM1141108: Nr70 danrer skin 24months rep1; Danio rerio; RNA Seq,GSM1141108 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141108,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr70_2y.fastq.gz,fastq,2239190508.0,29463033.0,GSM1141108 r1,0:76,A:599665392;C:514113743;G:531871299;T:592730477;N:809597,76,,,,599665392,514113743,531871299,592730477,809597,SRX277685,SRS420785,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.89167,,0.09031,,0.73515,,0.49129,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36752,SRR850597,SRX277684,SRS420784,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr30 danrer skin 5months rep9,GSM1141107,,source name:total RNA extracted from skin|strain:TuAB|age:5 month|tissue:skin,Nr30 danrer skin 5months rep9,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:5 month|tissue:skin,GSM1141107,GSM1141107: Nr30 danrer skin 5months rep9; Danio rerio; RNA Seq,GSM1141107 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141107,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr30_5m.fastq.gz,fastq,2502725752.0,32930602.0,GSM1141107 r1,0:76,A:684745428;C:570860380;G:565883575;T:679750149;N:1486220,76,,,,684745428,570860380,565883575,679750149,1486220,SRX277684,SRS420784,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91789,,0.10423,,0.7263,,0.4909,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36753,SRR850596,SRX277683,SRS420783,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr29 danrer skin 5months rep8,GSM1141106,,source name:total RNA extracted from skin|strain:TuAB|age:5 month|tissue:skin,Nr29 danrer skin 5months rep8,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:5 month|tissue:skin,GSM1141106,GSM1141106: Nr29 danrer skin 5months rep8; Danio rerio; RNA Seq,GSM1141106 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141106,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr29_5m.fastq.gz,fastq,2012479468.0,26479993.0,GSM1141106 r1,0:76,A:558800574;C:450898608;G:447907004;T:553773824;N:1099458,76,,,,558800574,450898608,447907004,553773824,1099458,SRX277683,SRS420783,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.9055,,0.1197,,0.7119,,0.48553,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36754,SRR850595,SRX277682,SRS420782,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr28 danrer skin 5months rep7,GSM1141105,,source name:total RNA extracted from skin|strain:TuAB|age:5 month|tissue:skin,Nr28 danrer skin 5months rep7,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:5 month|tissue:skin,GSM1141105,GSM1141105: Nr28 danrer skin 5months rep7; Danio rerio; RNA Seq,GSM1141105 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141105,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr28_5m.fastq.gz,fastq,2075098224.0,27303924.0,GSM1141105 r1,0:76,A:555062827;C:487427075;G:487360186;T:544381395;N:866741,76,,,,555062827,487427075,487360186,544381395,866741,SRX277682,SRS420782,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91784,,0.08866,,0.73208,,0.47793,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36755,SRR850594,SRX277681,SRS420781,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr27 danrer skin 5months rep6,GSM1141104,,source name:total RNA extracted from skin|strain:TuAB|age:5 month|tissue:skin,Nr27 danrer skin 5months rep6,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:5 month|tissue:skin,GSM1141104,GSM1141104: Nr27 danrer skin 5months rep6; Danio rerio; RNA Seq,GSM1141104 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141104,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr27_5m.fastq.gz,fastq,2415354480.0,31780980.0,GSM1141104 r1,0:76,A:661012615;C:552471296;G:546304105;T:654315033;N:1251431,76,,,,661012615,552471296,546304105,654315033,1251431,SRX277681,SRS420781,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91282,,0.104,,0.7236,,0.48186,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36756,SRR850593,SRX277680,SRS420780,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr26 danrer skin 5months rep5,GSM1141103,,source name:total RNA extracted from skin|strain:TuAB|age:5 month|tissue:skin,Nr26 danrer skin 5months rep5,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:5 month|tissue:skin,GSM1141103,GSM1141103: Nr26 danrer skin 5months rep5; Danio rerio; RNA Seq,GSM1141103 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141103,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr26_5m_l8_export.fq.gz,fastq,1866135464.0,24554414.0,GSM1141103 r1,0:76,A:495417130;C:442746432;G:436942898;T:490685510;N:343494,76,,,,495417130,442746432,436942898,490685510,343494,SRX277680,SRS420780,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92443,,0.08023,,0.7288,,0.48902,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36757,SRR850592,SRX277679,SRS420778,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr25 danrer skin 5months rep4,GSM1141102,,source name:total RNA extracted from skin|strain:TuAB|age:5 month|tissue:skin,Nr25 danrer skin 5months rep4,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:5 month|tissue:skin,GSM1141102,GSM1141102: Nr25 danrer skin 5months rep4; Danio rerio; RNA Seq,GSM1141102 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141102,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr25_5m_l7_export.fq.gz,fastq,2146470508.0,28243033.0,GSM1141102 r1,0:76,A:583770580;C:491677255;G:491536604;T:579365742;N:120327,76,,,,583770580,491677255,491536604,579365742,120327,SRX277679,SRS420778,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.89568,,0.11303,,0.71778,,0.47882,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36758,SRR850591,SRX277678,SRS420779,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr23 danrer skin 5months rep3,GSM1141101,,source name:total RNA extracted from skin|strain:TuAB|age:5 month|tissue:skin,Nr23 danrer skin 5months rep3,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:5 month|tissue:skin,GSM1141101,GSM1141101: Nr23 danrer skin 5months rep3; Danio rerio; RNA Seq,GSM1141101 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141101,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr23_5m_l6_export.fq.gz,fastq,2628691268.0,34588043.0,GSM1141101 r1,0:76,A:693944705;C:617538873;G:611502862;T:705582669;N:122159,76,,,,693944705,617538873,611502862,705582669,122159,SRX277678,SRS420779,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92203,,0.09747,,0.71378,,0.47796,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36759,SRR850590,SRX277677,SRS420777,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr22 danrer skin 5months rep2,GSM1141100,,source name:total RNA extracted from skin|strain:TuAB|age:5 month|tissue:skin,Nr22 danrer skin 5months rep2,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:5 month|tissue:skin,GSM1141100,GSM1141100: Nr22 danrer skin 5months rep2; Danio rerio; RNA Seq,GSM1141100 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141100,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr22_5m.fastq.gz,fastq,3561382724.0,46860299.0,GSM1141100 r1,0:76,A:956105654;C:826498432;G:826128735;T:952426883;N:223020,76,,,,956105654,826498432,826128735,952426883,223020,SRX277677,SRS420777,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93192,,0.09055,,0.72876,,0.52416,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36760,SRR850589,SRX277676,SRS420776,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr21 danrer skin 5months rep1,GSM1141099,,source name:total RNA extracted from skin|strain:TuAB|age:5 month|tissue:skin,Nr21 danrer skin 5months rep1,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:5 month|tissue:skin,GSM1141099,GSM1141099: Nr21 danrer skin 5months rep1; Danio rerio; RNA Seq,GSM1141099 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141099,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr21_5m_l4_export.fq.gz,fastq,2709932304.0,35657004.0,GSM1141099 r1,0:76,A:730073083;C:631072920;G:619401848;T:729203191;N:181262,76,,,,730073083,631072920,619401848,729203191,181262,SRX277676,SRS420776,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92839,,0.10379,,0.71392,,0.4865,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>40</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>40</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>47</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>40</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>40</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,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