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
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
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
38389,SRR1821850,SRX893451,SRS859516,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin208 36m 3.75nM roten1 8w rep6,GSM1620997,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,skin208 36m 3.75nM roten1 8w rep6,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,GSM1620997,GSM1620997: skin208 36m 3.75nM roten1 8w rep6; Danio rerio; RNA Seq,GSM1620997,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620997,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,208_skin.fastq.gz,fastq,1960697700.0,39213954.0,GSM1620997 r1,0:50,A:532792887;C:452528560;G:441240826;T:533522462;N:612965,50,,,,532792887,452528560,441240826,533522462,612965,SRX893451,SRS859516,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92216,,0.09326,,0.72147,,0.49795,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38390,SRR1821849,SRX893450,SRS859517,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin207 36m 3.75nM roten1 8w rep5,GSM1620996,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,skin207 36m 3.75nM roten1 8w rep5,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,GSM1620996,GSM1620996: skin207 36m 3.75nM roten1 8w rep5; Danio rerio; RNA Seq,GSM1620996,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620996,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,207_skin.fastq.gz,fastq,1644999850.0,32899997.0,GSM1620996 r1,0:50,A:444439104;C:381379643;G:371782441;T:447032625;N:366037,50,,,,444439104,381379643,371782441,447032625,366037,SRX893450,SRS859517,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.90733,,0.0971,,0.71518,,0.48175,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38391,SRR1821848,SRX893449,SRS859518,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin206 36m 3.75nM roten1 8w rep4,GSM1620995,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,skin206 36m 3.75nM roten1 8w rep4,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,GSM1620995,GSM1620995: skin206 36m 3.75nM roten1 8w rep4; Danio rerio; RNA Seq,GSM1620995,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620995,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,206_skin.fastq.gz,fastq,2333175800.0,46663516.0,GSM1620995 r1,0:50,A:629018600;C:541489412;G:532198591;T:629985712;N:483485,50,,,,629018600,541489412,532198591,629985712,483485,SRX893449,SRS859518,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91718,,0.10454,,0.70974,,0.47753,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38392,SRR1821847,SRX893448,SRS859519,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin205 36m 3.75nM roten1 8w rep3,GSM1620994,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,skin205 36m 3.75nM roten1 8w rep3,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,GSM1620994,GSM1620994: skin205 36m 3.75nM roten1 8w rep3; Danio rerio; RNA Seq,GSM1620994,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620994,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,205_skin.fastq.gz,fastq,1885120700.0,37702414.0,GSM1620994 r1,0:50,A:509247143;C:431669478;G:422352918;T:511569209;N:10281952,50,,,,509247143,431669478,422352918,511569209,10281952,SRX893448,SRS859519,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.90066,,0.107,,0.71415,,0.48729,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38393,SRR1821846,SRX893447,SRS859520,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin204 36m 3.75nM roten1 8w rep2,GSM1620993,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,skin204 36m 3.75nM roten1 8w rep2,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,GSM1620993,GSM1620993: skin204 36m 3.75nM roten1 8w rep2; Danio rerio; RNA Seq,GSM1620993,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620993,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,204_skin.fastq.gz,fastq,2039214400.0,40784288.0,GSM1620993 r1,0:50,A:554630438;C:468936669;G:460030131;T:555276071;N:341091,50,,,,554630438,468936669,460030131,555276071,341091,SRX893447,SRS859520,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91261,,0.11181,,0.70832,,0.4889,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38394,SRR1821845,SRX893446,SRS859521,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin203 36m 3.75nM roten1 8w rep1,GSM1620992,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,skin203 36m 3.75nM roten1 8w rep1,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:3.75nM roten1|duration:8 weeks,GSM1620992,GSM1620992: skin203 36m 3.75nM roten1 8w rep1; Danio rerio; RNA Seq,GSM1620992,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620992,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,203_skin.fastq.gz,fastq,2169512850.0,43390257.0,GSM1620992 r1,0:50,A:583644936;C:503133232;G:492417551;T:585152349;N:5164782,50,,,,583644936,503133232,492417551,585152349,5164782,SRX893446,SRS859521,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91263,,0.1004,,0.71376,,0.48566,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38395,SRR1821844,SRX893445,SRS859522,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin172 36m 3.75nM roten1 3w rep5,GSM1620991,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:3.75nM roten1|duration:3 weeks,skin172 36m 3.75nM roten1 3w rep5,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:3.75nM roten1|duration:3 weeks,GSM1620991,GSM1620991: skin172 36m 3.75nM roten1 3w rep5; Danio rerio; RNA Seq,GSM1620991,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620991,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,172_skin.fastq.gz,fastq,1922415800.0,38448316.0,GSM1620991 r1,0:50,A:517025440;C:447202733;G:438389966;T:519578929;N:218732,50,,,,517025440,447202733,438389966,519578929,218732,SRX893445,SRS859522,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92509,,0.08635,,0.71747,,0.48682,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38396,SRR1821843,SRX893444,SRS859523,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin171 36m 3.75nM roten1 3w rep4,GSM1620990,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:3.75nM roten1|duration:3 weeks,skin171 36m 3.75nM roten1 3w rep4,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:3.75nM roten1|duration:3 weeks,GSM1620990,GSM1620990: skin171 36m 3.75nM roten1 3w rep4; Danio rerio; RNA Seq,GSM1620990,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620990,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,171_skin.fastq.gz,fastq,1795802450.0,35916049.0,GSM1620990 r1,0:50,A:482833890;C:415521773;G:409072097;T:485286706;N:3087984,50,,,,482833890,415521773,409072097,485286706,3087984,SRX893444,SRS859523,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91548,,0.08856,,0.71153,,0.48407,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38397,SRR1821842,SRX893443,SRS859524,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin170 36m 3.75nM roten1 3w rep3,GSM1620989,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:3.75nM roten1|duration:3 weeks,skin170 36m 3.75nM roten1 3w rep3,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:3.75nM roten1|duration:3 weeks,GSM1620989,GSM1620989: skin170 36m 3.75nM roten1 3w rep3; Danio rerio; RNA Seq,GSM1620989,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620989,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,170_skin.fastq.gz,fastq,1844753700.0,36895074.0,GSM1620989 r1,0:50,A:496864420;C:428934827;G:420432768;T:498397505;N:124180,50,,,,496864420,428934827,420432768,498397505,124180,SRX893443,SRS859524,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92475,,0.08478,,0.71123,,0.5082,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38398,SRR1821841,SRX893442,SRS859525,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin169 36m 3.75nM roten1 3w rep2,GSM1620988,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:3.75nM roten1|duration:3 weeks,skin169 36m 3.75nM roten1 3w rep2,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:3.75nM roten1|duration:3 weeks,GSM1620988,GSM1620988: skin169 36m 3.75nM roten1 3w rep2; Danio rerio; RNA Seq,GSM1620988,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620988,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,169_skin.fastq.gz,fastq,1832979850.0,36659597.0,GSM1620988 r1,0:50,A:492012429;C:429700408;G:417026859;T:494015583;N:224571,50,,,,492012429,429700408,417026859,494015583,224571,SRX893442,SRS859525,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93738,,0.07334,,0.71936,,0.5123,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38399,SRR1821840,SRX893441,SRS859526,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin168 36m 3.75nM roten1 3w rep1,GSM1620987,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:3.75nM roten1|duration:3 weeks,skin168 36m 3.75nM roten1 3w rep1,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:3.75nM roten1|duration:3 weeks,GSM1620987,GSM1620987: skin168 36m 3.75nM roten1 3w rep1; Danio rerio; RNA Seq,GSM1620987,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620987,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,168_skin.fastq.gz,fastq,1811602600.0,36232052.0,GSM1620987 r1,0:50,A:482632140;C:422445561;G:412836586;T:485392445;N:8295868,50,,,,482632140,422445561,412836586,485392445,8295868,SRX893441,SRS859526,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.9185,,0.07569,,0.71368,,0.49409,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38400,SRR1821839,SRX893440,SRS859527,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin188 36m control 8w rep4,GSM1620986,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:n1|duration:8 weeks,skin188 36m control 8w rep4,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:n1|duration:8 weeks,GSM1620986,GSM1620986: skin188 36m control 8w rep4; Danio rerio; RNA Seq,GSM1620986,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620986,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,188_skin.fastq.gz,fastq,1797310350.0,35946207.0,GSM1620986 r1,0:50,A:486641633;C:413978398;G:404993329;T:489015902;N:2681088,50,,,,486641633,413978398,404993329,489015902,2681088,SRX893440,SRS859527,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91594,,0.09158,,0.72082,,0.51585,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38401,SRR1821838,SRX893439,SRS859528,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin187 36m control 8w rep3,GSM1620985,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:n1|duration:8 weeks,skin187 36m control 8w rep3,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:n1|duration:8 weeks,GSM1620985,GSM1620985: skin187 36m control 8w rep3; Danio rerio; RNA Seq,GSM1620985,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620985,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,187_skin.fastq.gz,fastq,1951002950.0,39020059.0,GSM1620985 r1,0:50,A:520701869;C:457907686;G:446812256;T:524196019;N:1385120,50,,,,520701869,457907686,446812256,524196019,1385120,SRX893439,SRS859528,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.9211,,0.07622,,0.71145,,0.46944,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38402,SRR1821837,SRX893438,SRS859529,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin186 36m control 8w rep2,GSM1620984,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:n1|duration:8 weeks,skin186 36m control 8w rep2,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:n1|duration:8 weeks,GSM1620984,GSM1620984: skin186 36m control 8w rep2; Danio rerio; RNA Seq,GSM1620984,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620984,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,186_skin.fastq.gz,fastq,2016117600.0,40322352.0,GSM1620984 r1,0:50,A:545084574;C:465634059;G:454024180;T:546321124;N:5053663,50,,,,545084574,465634059,454024180,546321124,5053663,SRX893438,SRS859529,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91606,,0.09659,,0.71246,,0.52973,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38403,SRR1821836,SRX893437,SRS859530,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin185 36m control 8w rep1,GSM1620983,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:n1|duration:8 weeks,skin185 36m control 8w rep1,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:n1|duration:8 weeks,GSM1620983,GSM1620983: skin185 36m control 8w rep1; Danio rerio; RNA Seq,GSM1620983,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620983,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,185_skin.fastq.gz,fastq,1862747450.0,37254949.0,GSM1620983 r1,0:50,A:506321471;C:429322966;G:419158880;T:507760233;N:183900,50,,,,506321471,429322966,419158880,507760233,183900,SRX893437,SRS859530,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.9187,,0.09926,,0.70755,,0.51593,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38404,SRR1821835,SRX893436,SRS859531,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin162 36m control 3w rep3,GSM1620982,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:n1|duration:3 weeks,skin162 36m control 3w rep3,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:n1|duration:3 weeks,GSM1620982,GSM1620982: skin162 36m control 3w rep3; Danio rerio; RNA Seq,GSM1620982,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620982,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,162_skin.fastq.gz,fastq,2058728600.0,41174572.0,GSM1620982 r1,0:50,A:545731040;C:487775330;G:479420254;T:545686109;N:115867,50,,,,545731040,487775330,479420254,545686109,115867,SRX893436,SRS859531,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93508,,0.07365,,0.70688,,0.4827,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38405,SRR1821834,SRX893435,SRS859532,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin161 36m control 3w rep2,GSM1620981,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:n1|duration:3 weeks,skin161 36m control 3w rep2,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:n1|duration:3 weeks,GSM1620981,GSM1620981: skin161 36m control 3w rep2; Danio rerio; RNA Seq,GSM1620981,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620981,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,161_skin.fastq.gz,fastq,1897142450.0,37942849.0,GSM1620981 r1,0:50,A:508043218;C:440251400;G:428510637;T:511046395;N:9290800,50,,,,508043218,440251400,428510637,511046395,9290800,SRX893435,SRS859532,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.90644,,0.0826,,0.71358,,0.49573,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38406,SRR1821833,SRX893434,SRS859533,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin160 36m control 3w rep1,GSM1620980,,source name:total RNA extracted from skin|tissue:skin|age:36 month|treatment:n1|duration:3 weeks,skin160 36m control 3w rep1,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:36 month|treatment:n1|duration:3 weeks,GSM1620980,GSM1620980: skin160 36m control 3w rep1; Danio rerio; RNA Seq,GSM1620980,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620980,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,160_skin.fastq.gz,fastq,1829692650.0,36593853.0,GSM1620980 r1,0:50,A:493906226;C:425214476;G:414775189;T:495470406;N:326353,50,,,,493906226,425214476,414775189,495470406,326353,SRX893434,SRS859533,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92036,,0.09412,,0.71484,,0.48928,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38407,SRR1821832,SRX893433,SRS859534,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin184 12m control 8w rep3,GSM1620979,,source name:total RNA extracted from skin|tissue:skin|age:12 month|treatment:n1|duration:8 weeks,skin184 12m control 8w rep3,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:12 month|treatment:n1|duration:8 weeks,GSM1620979,GSM1620979: skin184 12m control 8w rep3; Danio rerio; RNA Seq,GSM1620979,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620979,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,184_skin.fastq.gz,fastq,1888865600.0,37777312.0,GSM1620979 r1,0:50,A:510384459;C:436312051;G:428261723;T:512241273;N:1666094,50,,,,510384459,436312051,428261723,512241273,1666094,SRX893433,SRS859534,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91542,,0.0997,,0.70082,,0.49633,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38408,SRR1821831,SRX893432,SRS859535,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin183 12m control 8w rep2,GSM1620978,,source name:total RNA extracted from skin|tissue:skin|age:12 month|treatment:n1|duration:8 weeks,skin183 12m control 8w rep2,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:12 month|treatment:n1|duration:8 weeks,GSM1620978,GSM1620978: skin183 12m control 8w rep2; Danio rerio; RNA Seq,GSM1620978,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620978,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,183_skin.fastq.gz,fastq,2011969000.0,40239380.0,GSM1620978 r1,0:50,A:541080523;C:469282719;G:455255702;T:544440274;N:1909782,50,,,,541080523,469282719,455255702,544440274,1909782,SRX893432,SRS859535,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92404,,0.104,,0.70619,,0.47963,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38409,SRR1821830,SRX893431,SRS859536,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin182 12m control 8w rep1,GSM1620977,,source name:total RNA extracted from skin|tissue:skin|age:12 month|treatment:n1|duration:8 weeks,skin182 12m control 8w rep1,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:12 month|treatment:n1|duration:8 weeks,GSM1620977,GSM1620977: skin182 12m control 8w rep1; Danio rerio; RNA Seq,GSM1620977,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620977,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,182_skin.fastq.gz,fastq,1974072600.0,39481452.0,GSM1620977 r1,0:50,A:532294515;C:457485293;G:447416004;T:532212696;N:4664092,50,,,,532294515,457485293,447416004,532212696,4664092,SRX893431,SRS859536,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91699,,0.10118,,0.71163,,0.49086,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38410,SRR1821829,SRX893430,SRS859537,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin159 12m control 3w rep3,GSM1620976,,source name:total RNA extracted from skin|tissue:skin|age:12 month|treatment:n1|duration:3 weeks,skin159 12m control 3w rep3,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:12 month|treatment:n1|duration:3 weeks,GSM1620976,GSM1620976: skin159 12m control 3w rep3; Danio rerio; RNA Seq,GSM1620976,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620976,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,159_skin.fastq.gz,fastq,1887853800.0,37757076.0,GSM1620976 r1,0:50,A:510759018;C:434865500;G:426519929;T:512499569;N:3209784,50,,,,510759018,434865500,426519929,512499569,3209784,SRX893430,SRS859537,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91654,,0.1023,,0.70607,,0.49388,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38411,SRR1821828,SRX893429,SRS859538,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin158 12m control 3w rep2,GSM1620975,,source name:total RNA extracted from skin|tissue:skin|age:12 month|treatment:n1|duration:3 weeks,skin158 12m control 3w rep2,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:12 month|treatment:n1|duration:3 weeks,GSM1620975,GSM1620975: skin158 12m control 3w rep2; Danio rerio; RNA Seq,GSM1620975,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620975,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,158_skin.fastq.gz,fastq,1818559300.0,36371186.0,GSM1620975 r1,0:50,A:487547586;C:425039144;G:415674047;T:490148749;N:149774,50,,,,487547586,425039144,415674047,490148749,149774,SRX893429,SRS859538,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93033,,0.09871,,0.66103,,0.48246,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
38412,SRR1821827,SRX893428,SRS859539,SRP055573,PRJNA276667,RNA seq of zebrafish brain  liver and skin during perturbation with rotenone at young and old age,GSE66362,Transcriptome Analysis,Zebrafish of two different age groups 12 month and 36 month were treated with low amounts of rotenone mild stress and compared to untreated zebrafish. Two different durations were used 3 and 8 weeks. Illumina sequencing HiSeq2000 was applied to generate 50bp single end reads. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 68 sample: 3 tissues brain  liver  skin; 2 age groups 12 month and 36 month; controls and rotenone treated samples; 2 6 biological replicates for each group,,pubmed:27135165,,skin157 12m control 3w rep1,GSM1620974,,source name:total RNA extracted from skin|tissue:skin|age:12 month|treatment:n1|duration:3 weeks,skin157 12m control 3w rep1,FASTQ files were extracted using Illumina Casava software v1.8.2.,total RNA extracted from skin,Tanks with male zebrafish were supplemented with 3.75 nM rotenone or DMSO control for 3 weeks and  8 weeks  respectively. 50% tank water was changed twice a week and fresh Rotenone and DMSO was added to the tanks.,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,Zebrafish Danio rerio were raised under standard conditions.,tissue:skin|age:12 month|treatment:n1|duration:3 weeks,GSM1620974,GSM1620974: skin157 12m control 3w rep1; Danio rerio; RNA Seq,GSM1620974,,1,Total RNA was isolated using QIAzol Qiagen  Hilden  Germany based on the phenol/chloroform extraction method. postwards the RNA was quantified photometrically with a NanoDrop 1000 PeqLab  Erlangen  Germany and stored at  80 °C until use. For library preparation an amount of around 2.5 ug of total RNA per sample was processed using Illumina's TruSeqTM RNA Sample Prep Kit Illumina; San Diego; CA  USA following the manufacturer's instruction.,GEO Accession:GSM1620974,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP055573,,,157_skin.fastq.gz,fastq,1855321100.0,37106422.0,GSM1620974 r1,0:50,A:495304825;C:434601244;G:425046390;T:498975673;N:1392968,50,,,,495304825,434601244,425046390,498975673,1392968,SRX893428,SRS859539,SRA244883,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91768,,0.09102,,0.7012,,0.49299,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2015-02-27,Adult,Adult,Skin,Surface Structure
40001,SRR2751044,SRX1362114,SRS1125416,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 111,GSM1915550,,source name:total RNA extracted from skin|strain:AB JxTu Tgwt1a:GFP|age:42 month|age category:old 2|tissue:skin,NH FLI skin 111,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgwt1a:GFP|age:42 month|age category:old 2|tissue:skin,GSM1915550,GSM1915550: NH FLI skin 111; Danio rerio; RNA Seq,GSM1915550,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915550,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_42m_DR111.fq.gz,fastq,3159101450.0,63182029.0,GSM1915550 r1,0:50,A:848692326;C:737950616;G:722461308;T:849228405;N:768795,50,,,,848692326,737950616,722461308,849228405,768795,SRX1362114,SRS1125416,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.9327,,0.09435,,0.70737,,0.50049,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40002,SRR2751043,SRX1362113,SRS1125415,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 109,GSM1915549,,source name:total RNA extracted from skin|strain:AB JxTu Tgwt1a:GFP|age:42 month|age category:old 2|tissue:skin,NH FLI skin 109,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgwt1a:GFP|age:42 month|age category:old 2|tissue:skin,GSM1915549,GSM1915549: NH FLI skin 109; Danio rerio; RNA Seq,GSM1915549,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915549,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_42m_DR109.fq.gz,fastq,2638118700.0,52762374.0,GSM1915549 r1,0:50,A:699567604;C:623885297;G:610871040;T:703405737;N:389022,50,,,,699567604,623885297,610871040,703405737,389022,SRX1362113,SRS1125415,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93453,,0.09144,,0.7191,,0.47437,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40003,SRR2751042,SRX1362112,SRS1125418,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 108,GSM1915548,,source name:total RNA extracted from skin|strain:AB JxTu Tgwt1a:GFP|age:42 month|age category:old 2|tissue:skin,NH FLI skin 108,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgwt1a:GFP|age:42 month|age category:old 2|tissue:skin,GSM1915548,GSM1915548: NH FLI skin 108; Danio rerio; RNA Seq,GSM1915548,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915548,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_42m_DR108.fq.gz,fastq,2354900150.0,47098003.0,GSM1915548 r1,0:50,A:629252405;C:552452722;G:540951538;T:632145411;N:98074,50,,,,629252405,552452722,540951538,632145411,98074,SRX1362112,SRS1125418,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93959,,0.09384,,0.71764,,0.49807,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40004,SRR2751041,SRX1362111,SRS1125419,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 107,GSM1915547,,source name:total RNA extracted from skin|strain:AB JxTu Tgwt1a:GFP|age:42 month|age category:old 2|tissue:skin,NH FLI skin 107,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgwt1a:GFP|age:42 month|age category:old 2|tissue:skin,GSM1915547,GSM1915547: NH FLI skin 107; Danio rerio; RNA Seq,GSM1915547,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915547,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_42m_DR107.fq.gz,fastq,2518777200.0,50375544.0,GSM1915547 r1,0:50,A:678501999;C:582249482;G:579224840;T:678502233;N:298646,50,,,,678501999,582249482,579224840,678502233,298646,SRX1362111,SRS1125419,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.9366,,0.10967,,0.71977,,0.4795,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40005,SRR2751040,SRX1362110,SRS1125417,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 106,GSM1915546,,source name:total RNA extracted from skin|strain:AB JxTu Tgwt1a:GFP|age:42 month|age category:old 2|tissue:skin,NH FLI skin 106,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgwt1a:GFP|age:42 month|age category:old 2|tissue:skin,GSM1915546,GSM1915546: NH FLI skin 106; Danio rerio; RNA Seq,GSM1915546,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915546,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_42m_DR106.fq.gz,fastq,2372061450.0,47441229.0,GSM1915546 r1,0:50,A:644690580;C:546357177;G:533675326;T:646884182;N:454185,50,,,,644690580,546357177,533675326,646884182,454185,SRX1362110,SRS1125417,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92831,,0.10593,,0.70453,,0.49444,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40006,SRR2751039,SRX1362109,SRS1125433,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 150,GSM1915545,,source name:total RNA extracted from skin|strain:AB JxTu Tgpu.1:GFP|age:36 month|age category:old 1|tissue:skin,NH FLI skin 150,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgpu.1:GFP|age:36 month|age category:old 1|tissue:skin,GSM1915545,GSM1915545: NH FLI skin 150; Danio rerio; RNA Seq,GSM1915545,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915545,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_36m_DR150.fq.gz,fastq,2518891400.0,50377828.0,GSM1915545 r1,0:50,A:674241151;C:589651596;G:577346091;T:677408422;N:244140,50,,,,674241151,589651596,577346091,677408422,244140,SRX1362109,SRS1125433,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92457,,0.09542,,0.72985,,0.47778,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40007,SRR2751038,SRX1362108,SRS1125421,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 149,GSM1915544,,source name:total RNA extracted from skin|strain:AB JxTu Tgpu.1:GFP|age:36 month|age category:old 1|tissue:skin,NH FLI skin 149,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgpu.1:GFP|age:36 month|age category:old 1|tissue:skin,GSM1915544,GSM1915544: NH FLI skin 149; Danio rerio; RNA Seq,GSM1915544,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915544,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_36m_DR149.fq.gz,fastq,2379227850.0,47584557.0,GSM1915544 r1,0:50,A:637191017;C:555983414;G:544406685;T:641356487;N:290247,50,,,,637191017,555983414,544406685,641356487,290247,SRX1362108,SRS1125421,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92532,,0.09729,,0.72813,,0.46517,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40008,SRR2751037,SRX1362107,SRS1125420,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 147,GSM1915543,,source name:total RNA extracted from skin|strain:AB JxTu Tgpu.1:GFP|age:36 month|age category:old 1|tissue:skin,NH FLI skin 147,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgpu.1:GFP|age:36 month|age category:old 1|tissue:skin,GSM1915543,GSM1915543: NH FLI skin 147; Danio rerio; RNA Seq,GSM1915543,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915543,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_36m_DR147.fq.gz,fastq,2551716800.0,51034336.0,GSM1915543 r1,0:50,A:678806838;C:599057712;G:596701555;T:677051257;N:99438,50,,,,678806838,599057712,596701555,677051257,99438,SRX1362107,SRS1125420,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92591,,0.09543,,0.72648,,0.45136,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40009,SRR2751036,SRX1362106,SRS1125422,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 146,GSM1915542,,source name:total RNA extracted from skin|strain:AB JxTu Tgpu.1:GFP|age:36 month|age category:old 1|tissue:skin,NH FLI skin 146,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgpu.1:GFP|age:36 month|age category:old 1|tissue:skin,GSM1915542,GSM1915542: NH FLI skin 146; Danio rerio; RNA Seq,GSM1915542,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915542,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_36m_DR146.fq.gz,fastq,2471171650.0,49423433.0,GSM1915542 r1,0:50,A:660642892;C:581474319;G:564732545;T:664036504;N:285390,50,,,,660642892,581474319,564732545,664036504,285390,SRX1362106,SRS1125422,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93248,,0.09495,,0.72285,,0.47165,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40010,SRR2751035,SRX1362105,SRS1125411,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 145,GSM1915541,,source name:total RNA extracted from skin|strain:AB JxTu Tgpu.1:GFP|age:36 month|age category:old 1|tissue:skin,NH FLI skin 145,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgpu.1:GFP|age:36 month|age category:old 1|tissue:skin,GSM1915541,GSM1915541: NH FLI skin 145; Danio rerio; RNA Seq,GSM1915541,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915541,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_36m_DR145.fq.gz,fastq,2334774050.0,46695481.0,GSM1915541 r1,0:50,A:623200220;C:545447003;G:542943898;T:622762029;N:420900,50,,,,623200220,545447003,542943898,622762029,420900,SRX1362105,SRS1125411,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92775,,0.09711,,0.71898,,0.46519,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40011,SRR2751034,SRX1362104,SRS1125414,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 137,GSM1915540,,source name:total RNA extracted from skin|strain:AB JxTu|age:24 month|age category: |tissue:skin,NH FLI skin 137,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu|age:24 month|age category: |tissue:skin,GSM1915540,GSM1915540: NH FLI skin 137; Danio rerio; RNA Seq,GSM1915540,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915540,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_24m_DR137.fq.gz,fastq,2309179800.0,46183596.0,GSM1915540 r1,0:50,A:616966249;C:542043078;G:533727704;T:616305630;N:137139,50,,,,616966249,542043078,533727704,616305630,137139,SRX1362104,SRS1125414,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93876,,0.09313,,0.72358,,0.47709,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40012,SRR2751033,SRX1362103,SRS1125412,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 136,GSM1915539,,source name:total RNA extracted from skin|strain:AB JxTu|age:24 month|age category: |tissue:skin,NH FLI skin 136,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu|age:24 month|age category: |tissue:skin,GSM1915539,GSM1915539: NH FLI skin 136; Danio rerio; RNA Seq,GSM1915539,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915539,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_24m_DR136.fq.gz,fastq,2263743350.0,45274867.0,GSM1915539 r1,0:50,A:606303719;C:530430586;G:518717687;T:608118372;N:172986,50,,,,606303719,530430586,518717687,608118372,172986,SRX1362103,SRS1125412,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.9402,,0.0984,,0.71127,,0.47385,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40013,SRR2751032,SRX1362102,SRS1125425,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 135,GSM1915538,,source name:total RNA extracted from skin|strain:AB JxTu|age:24 month|age category: |tissue:skin,NH FLI skin 135,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu|age:24 month|age category: |tissue:skin,GSM1915538,GSM1915538: NH FLI skin 135; Danio rerio; RNA Seq,GSM1915538,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915538,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_24m_DR135.fq.gz,fastq,2314971400.0,46299428.0,GSM1915538 r1,0:50,A:623966126;C:535953629;G:528078617;T:626790067;N:182961,50,,,,623966126,535953629,528078617,626790067,182961,SRX1362102,SRS1125425,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.94208,,0.09788,,0.72381,,0.50181,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40014,SRR2751031,SRX1362101,SRS1125423,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 134,GSM1915537,,source name:total RNA extracted from skin|strain:AB JxTu|age:24 month|age category: |tissue:skin,NH FLI skin 134,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu|age:24 month|age category: |tissue:skin,GSM1915537,GSM1915537: NH FLI skin 134; Danio rerio; RNA Seq,GSM1915537,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915537,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_24m_DR134.fq.gz,fastq,2308809950.0,46176199.0,GSM1915537 r1,0:50,A:616608249;C:543194614;G:529954288;T:618726585;N:326214,50,,,,616608249,543194614,529954288,618726585,326214,SRX1362101,SRS1125423,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93418,,0.09164,,0.7175,,0.46791,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40015,SRR2751030,SRX1362100,SRS1125424,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 133,GSM1915536,,source name:total RNA extracted from skin|strain:AB JxTu|age:24 month|age category: |tissue:skin,NH FLI skin 133,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu|age:24 month|age category: |tissue:skin,GSM1915536,GSM1915536: NH FLI skin 133; Danio rerio; RNA Seq,GSM1915536,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915536,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_24m_DR133.fq.gz,fastq,2297522100.0,45950442.0,GSM1915536 r1,0:50,A:613565891;C:538631563;G:528986620;T:616053581;N:284445,50,,,,613565891,538631563,528986620,616053581,284445,SRX1362100,SRS1125424,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.9452,,0.095,,0.71104,,0.48911,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40016,SRR2751029,SRX1362099,SRS1125426,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 125,GSM1915535,,source name:total RNA extracted from skin|strain:AB JxTu Tgflk:mCherry|age:12 month|age category: |tissue:skin,NH FLI skin 125,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgflk:mCherry|age:12 month|age category: |tissue:skin,GSM1915535,GSM1915535: NH FLI skin 125; Danio rerio; RNA Seq,GSM1915535,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915535,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_12m_DR125.fq.gz,fastq,3302068200.0,66041364.0,GSM1915535 r1,0:50,A:884122674;C:774529608;G:756751070;T:886167108;N:497740,50,,,,884122674,774529608,756751070,886167108,497740,SRX1362099,SRS1125426,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93865,,0.08806,,0.72616,,0.51404,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40017,SRR2751028,SRX1362098,SRS1125427,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 122,GSM1915534,,source name:total RNA extracted from skin|strain:AB JxTu Tgflk:mCherry|age:12 month|age category: |tissue:skin,NH FLI skin 122,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgflk:mCherry|age:12 month|age category: |tissue:skin,GSM1915534,GSM1915534: NH FLI skin 122; Danio rerio; RNA Seq,GSM1915534,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915534,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_12m_DR122.fq.gz,fastq,2377233850.0,47544677.0,GSM1915534 r1,0:50,A:642601261;C:551178683;G:540849985;T:642423287;N:180634,50,,,,642601261,551178683,540849985,642423287,180634,SRX1362098,SRS1125427,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93658,,0.08983,,0.72592,,0.51448,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40018,SRR2751027,SRX1362097,SRS1125428,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 121,GSM1915533,,source name:total RNA extracted from skin|strain:AB JxTu Tgflk:mCherry|age:12 month|age category: |tissue:skin,NH FLI skin 121,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgflk:mCherry|age:12 month|age category: |tissue:skin,GSM1915533,GSM1915533: NH FLI skin 121; Danio rerio; RNA Seq,GSM1915533,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915533,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_12m_DR121.fq.gz,fastq,2735944300.0,54718886.0,GSM1915533 r1,0:50,A:735622619;C:637792745;G:628439202;T:733845015;N:244719,50,,,,735622619,637792745,628439202,733845015,244719,SRX1362097,SRS1125428,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.942,,0.09314,,0.72356,,0.51726,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40019,SRR2751026,SRX1362096,SRS1125429,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 120,GSM1915532,,source name:total RNA extracted from skin|strain:AB JxTu Tgflk:mCherry|age:12 month|age category: |tissue:skin,NH FLI skin 120,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgflk:mCherry|age:12 month|age category: |tissue:skin,GSM1915532,GSM1915532: NH FLI skin 120; Danio rerio; RNA Seq,GSM1915532,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915532,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_12m_DR120.fq.gz,fastq,2372344950.0,47446899.0,GSM1915532 r1,0:50,A:638932034;C:550470960;G:548046506;T:634464502;N:430948,50,,,,638932034,550470960,548046506,634464502,430948,SRX1362096,SRS1125429,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93665,,0.0991,,0.7237,,0.49202,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40020,SRR2751025,SRX1362095,SRS1125430,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 119,GSM1915531,,source name:total RNA extracted from skin|strain:AB JxTu Tgflk:mCherry|age:12 month|age category: |tissue:skin,NH FLI skin 119,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu Tgflk:mCherry|age:12 month|age category: |tissue:skin,GSM1915531,GSM1915531: NH FLI skin 119; Danio rerio; RNA Seq,GSM1915531,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915531,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_12m_DR119.fq.gz,fastq,2618543150.0,52370863.0,GSM1915531 r1,0:50,A:708042520;C:607068819;G:594416518;T:708510167;N:505126,50,,,,708042520,607068819,594416518,708510167,505126,SRX1362095,SRS1125430,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93526,,0.0946,,0.71973,,0.50364,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40021,SRR2751024,SRX1362094,SRS1125431,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 98,GSM1915530,,source name:total RNA extracted from skin|strain:AB JxTu|age:6 month|age category:young|tissue:skin,NH FLI skin 98,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu|age:6 month|age category:young|tissue:skin,GSM1915530,GSM1915530: NH FLI skin 98; Danio rerio; RNA Seq,GSM1915530,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915530,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_6m_DR98.fq.gz,fastq,2547776750.0,50955535.0,GSM1915530 r1,0:50,A:683451413;C:596226761;G:580799071;T:687174232;N:125273,50,,,,683451413,596226761,580799071,687174232,125273,SRX1362094,SRS1125431,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93518,,0.09039,,0.72464,,0.50556,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40022,SRR2751023,SRX1362093,SRS1125432,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 96,GSM1915529,,source name:total RNA extracted from skin|strain:AB JxTu|age:6 month|age category:young|tissue:skin,NH FLI skin 96,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu|age:6 month|age category:young|tissue:skin,GSM1915529,GSM1915529: NH FLI skin 96; Danio rerio; RNA Seq,GSM1915529,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915529,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_6m_DR96.fq.gz,fastq,3475787450.0,69515749.0,GSM1915529 r1,0:50,A:934381912;C:816876197;G:800228290;T:923834043;N:467008,50,,,,934381912,816876197,800228290,923834043,467008,SRX1362093,SRS1125432,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92173,,0.09447,,0.76124,,0.49732,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40023,SRR2751022,SRX1362092,SRS1125434,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 94,GSM1915528,,source name:total RNA extracted from skin|strain:AB JxTu|age:6 month|age category:young|tissue:skin,NH FLI skin 94,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu|age:6 month|age category:young|tissue:skin,GSM1915528,GSM1915528: NH FLI skin 94; Danio rerio; RNA Seq,GSM1915528,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915528,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_6m_DR94.fq.gz,fastq,2342707850.0,46854157.0,GSM1915528 r1,0:50,A:627518600;C:548445446;G:537823256;T:628778691;N:141857,50,,,,627518600,548445446,537823256,628778691,141857,SRX1362092,SRS1125434,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.94121,,0.09466,,0.73089,,0.48547,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40024,SRR2751021,SRX1362091,SRS1125435,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 86,GSM1915527,,source name:total RNA extracted from skin|strain:AB JxTu|age:6 month|age category:young|tissue:skin,NH FLI skin 86,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu|age:6 month|age category:young|tissue:skin,GSM1915527,GSM1915527: NH FLI skin 86; Danio rerio; RNA Seq,GSM1915527,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915527,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_6m_DR86.fq.gz,fastq,2438130400.0,48762608.0,GSM1915527 r1,0:50,A:650474585;C:570893533;G:567553610;T:648755019;N:453653,50,,,,650474585,570893533,567553610,648755019,453653,SRX1362091,SRS1125435,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.94371,,0.09068,,0.72358,,0.49841,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
40025,SRR2751020,SRX1362090,SRS1125436,SRP065208,PRJNA299585,Deep sequencing of mRNA from Danio rerio at five different timepoints in three different tissues brain  liver  skin,GSE74244,Transcriptome Analysis,Comparison of temporal gene expression profiles Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 75 samples in sum; 5 age groups 6  12  24  36  42 month; 3 tissues brain  liver  skin; 5 samples per group,,pubmed:29382830,,NH FLI skin 85,GSM1915526,,source name:total RNA extracted from skin|strain:AB JxTu|age:6 month|age category:young|tissue:skin,NH FLI skin 85,Illumina Casava v1.8.0 software used for extraction of FASTQ files Reads were mapped using Tophat v2.0.6 to the genome taken the annotation into account:  tophat2  p 24  o OUTPUTDIR  g 1   no coverage search   transcriptome index=transcriptome data/Danio rerio.Zv9.73 Danio rerio.Zv9.73 Counting of reads per gene was done using htseq count: htseq count   quiet   stranded=no   idattr=gene id   mode=union SAMFILE Danio rerio.Zv9.73.gtf > OUTPUTFILE Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Gene were excluded from dataset if at least one sample has a RPKM of 0. Genome build: Ensemble Zv9.73 Supplementary files format and content: Excel file includes raw counts and RPKM values for each sample,total RNA extracted from skin,,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,,strain:AB JxTu|age:6 month|age category:young|tissue:skin,GSM1915526,GSM1915526: NH FLI skin 85; Danio rerio; RNA Seq,GSM1915526,,1,Total RNA from was extracted as described Baumgart et al.  2012; PMID:22487494 Preparation of libraries was done using Illumina’s TruSeq RNA sample prep kit following the manufacturer’s instruction.,GEO Accession:GSM1915526,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP065208,,,skin_6m_DR85.fq.gz,fastq,2599182950.0,51983659.0,GSM1915526 r1,0:50,A:698996178;C:606423487;G:594332434;T:699020747;N:410104,50,,,,698996178,606423487,594332434,699020747,410104,SRX1362090,SRS1125436,SRA306458,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93429,,0.10078,,0.70877,,0.4944,,50,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2015-10-21,Adult,Adult,Skin,Surface Structure
43247,SRR5921030,SRX3081857,SRS2420474,SRP115124,PRJNA397736,Sequencing of Danio rerio skin from wildtype and two tert mutant phenotypes  JenAge,GSE102434,Transcriptome Analysis,Comparison of gene expression profiles from Danio rerio skin of wiltype AB and homozygous/heterozygous tert mutant phenotypes AB tert /   AB tert+/. The RNA seq data comprises 3 groups. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: Overall Design: 13 samples in 3 groups: AB 5 samples  AB tert /  3 samples  AB tert+/  5 samples,,pubmed:34680427,,NH FLI 273 skin RNAseq,GSM2736513,,source name:skin|strain:AB tert / |tissue:skin|age:3 month|Sex:male,NH FLI 273 skin RNAseq,Sequence information was extracted in FastQ format using bcl2fastq software 1.8.4 data processing step. Reads were mapped using STAR 2.4.1d. Reads per gene were counted using featureCounts 1.4.6 p4. Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Genome build: genome: GRCz10  and respective gene annotation GRCz10.84 Supplementary files format and content: The Excel files include raw counts sample counts.xls and RPKM values sample rpkms.xls of all genes for each sample.,skin,,Extraction was done as described in Baumgart et al. 2012. PMID:22487494 Library preparation was done using Illumina's TruSeq RNA Library Preparation Kit v2 following the manufacturer's instruction.,,strain:AB tert / |tissue:skin|age:3 month|Sex:male,GSM2736513,GSM2736513: NH FLI 273 skin RNAseq; Danio rerio; RNA Seq,GSM2736513,,1,Extraction was done as described in Baumgart et al. 2012. PMID:22487494 Library preparation was done using Illumina's TruSeq RNA Library Preparation Kit v2 following the manufacturer's instruction.,GEO Accession:GSM2736513,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2500,,SRP115124,,,,,972558882.0,19069782.0,GSM2736513 r1,0:51,A:272729739;C:215238085;G:210941336;T:273588371;N:61351,51,,,,272729739,215238085,210941336,273588371,61351,SRX3081857,SRS2420474,SRA597914,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.95763,,0.05265,,0.76333,,0.66639,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2017-08-09,Adult,Adult,Skin,Surface Structure
43248,SRR5921029,SRX3081856,SRS2420473,SRP115124,PRJNA397736,Sequencing of Danio rerio skin from wildtype and two tert mutant phenotypes  JenAge,GSE102434,Transcriptome Analysis,Comparison of gene expression profiles from Danio rerio skin of wiltype AB and homozygous/heterozygous tert mutant phenotypes AB tert /   AB tert+/. The RNA seq data comprises 3 groups. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: Overall Design: 13 samples in 3 groups: AB 5 samples  AB tert /  3 samples  AB tert+/  5 samples,,pubmed:34680427,,NH FLI 271 skin RNAseq,GSM2736512,,source name:skin|strain:AB tert+/ |tissue:skin|age:3 month|Sex:male,NH FLI 271 skin RNAseq,Sequence information was extracted in FastQ format using bcl2fastq software 1.8.4 data processing step. Reads were mapped using STAR 2.4.1d. Reads per gene were counted using featureCounts 1.4.6 p4. Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Genome build: genome: GRCz10  and respective gene annotation GRCz10.84 Supplementary files format and content: The Excel files include raw counts sample counts.xls and RPKM values sample rpkms.xls of all genes for each sample.,skin,,Extraction was done as described in Baumgart et al. 2012. PMID:22487494 Library preparation was done using Illumina's TruSeq RNA Library Preparation Kit v2 following the manufacturer's instruction.,,strain:AB tert+/ |tissue:skin|age:3 month|Sex:male,GSM2736512,GSM2736512: NH FLI 271 skin RNAseq; Danio rerio; RNA Seq,GSM2736512,,1,Extraction was done as described in Baumgart et al. 2012. PMID:22487494 Library preparation was done using Illumina's TruSeq RNA Library Preparation Kit v2 following the manufacturer's instruction.,GEO Accession:GSM2736512,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2500,,SRP115124,,,,,1866408240.0,36596240.0,GSM2736512 r1,0:51,A:517174911;C:420938332;G:411043524;T:517150912;N:100561,51,,,,517174911,420938332,411043524,517150912,100561,SRX3081856,SRS2420473,SRA597914,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.94283,,0.07392,,0.7471,,0.61455,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2017-08-09,Adult,Adult,Skin,Surface Structure
43249,SRR5921028,SRX3081855,SRS2420475,SRP115124,PRJNA397736,Sequencing of Danio rerio skin from wildtype and two tert mutant phenotypes  JenAge,GSE102434,Transcriptome Analysis,Comparison of gene expression profiles from Danio rerio skin of wiltype AB and homozygous/heterozygous tert mutant phenotypes AB tert /   AB tert+/. The RNA seq data comprises 3 groups. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: Overall Design: 13 samples in 3 groups: AB 5 samples  AB tert /  3 samples  AB tert+/  5 samples,,pubmed:34680427,,NH FLI 270 skin RNAseq,GSM2736511,,source name:skin|strain:AB wildtype|tissue:skin|age:3 month|Sex:male,NH FLI 270 skin RNAseq,Sequence information was extracted in FastQ format using bcl2fastq software 1.8.4 data processing step. Reads were mapped using STAR 2.4.1d. Reads per gene were counted using featureCounts 1.4.6 p4. Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Genome build: genome: GRCz10  and respective gene annotation GRCz10.84 Supplementary files format and content: The Excel files include raw counts sample counts.xls and RPKM values sample rpkms.xls of all genes for each sample.,skin,,Extraction was done as described in Baumgart et al. 2012. PMID:22487494 Library preparation was done using Illumina's TruSeq RNA Library Preparation Kit v2 following the manufacturer's instruction.,,strain:AB wildtype|tissue:skin|age:3 month|Sex:male,GSM2736511,GSM2736511: NH FLI 270 skin RNAseq; Danio rerio; RNA Seq,GSM2736511,,1,Extraction was done as described in Baumgart et al. 2012. PMID:22487494 Library preparation was done using Illumina's TruSeq RNA Library Preparation Kit v2 following the manufacturer's instruction.,GEO Accession:GSM2736511,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2500,,SRP115124,,,,,1905337356.0,37359556.0,GSM2736511 r1,0:51,A:522553789;C:435303205;G:425434137;T:521931629;N:114596,51,,,,522553789,435303205,425434137,521931629,114596,SRX3081855,SRS2420475,SRA597914,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.94407,,0.07001,,0.74239,,0.59487,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2017-08-09,Adult,Adult,Skin,Surface Structure
43250,SRR5921027,SRX3081854,SRS2420476,SRP115124,PRJNA397736,Sequencing of Danio rerio skin from wildtype and two tert mutant phenotypes  JenAge,GSE102434,Transcriptome Analysis,Comparison of gene expression profiles from Danio rerio skin of wiltype AB and homozygous/heterozygous tert mutant phenotypes AB tert /   AB tert+/. The RNA seq data comprises 3 groups. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: Overall Design: 13 samples in 3 groups: AB 5 samples  AB tert /  3 samples  AB tert+/  5 samples,,pubmed:34680427,,NH FLI 264 skin RNAseq,GSM2736510,,source name:skin|strain:AB tert+/ |tissue:skin|age:3 month|Sex:male,NH FLI 264 skin RNAseq,Sequence information was extracted in FastQ format using bcl2fastq software 1.8.4 data processing step. Reads were mapped using STAR 2.4.1d. Reads per gene were counted using featureCounts 1.4.6 p4. Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Genome build: genome: GRCz10  and respective gene annotation GRCz10.84 Supplementary files format and content: The Excel files include raw counts sample counts.xls and RPKM values sample rpkms.xls of all genes for each sample.,skin,,Extraction was done as described in Baumgart et al. 2012. PMID:22487494 Library preparation was done using Illumina's TruSeq RNA Library Preparation Kit v2 following the manufacturer's instruction.,,strain:AB tert+/ |tissue:skin|age:3 month|Sex:male,GSM2736510,GSM2736510: NH FLI 264 skin RNAseq; Danio rerio; RNA Seq,GSM2736510,,1,Extraction was done as described in Baumgart et al. 2012. PMID:22487494 Library preparation was done using Illumina's TruSeq RNA Library Preparation Kit v2 following the manufacturer's instruction.,GEO Accession:GSM2736510,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2500,,SRP115124,,,,,2007950988.0,39371588.0,GSM2736510 r1,0:51,A:556985493;C:451429027;G:441126200;T:558303542;N:106726,51,,,,556985493,451429027,441126200,558303542,106726,SRX3081854,SRS2420476,SRA597914,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.94195,,0.07318,,0.74939,,0.62731,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2017-08-09,Adult,Adult,Skin,Surface Structure
43251,SRR5921026,SRX3081853,SRS2420477,SRP115124,PRJNA397736,Sequencing of Danio rerio skin from wildtype and two tert mutant phenotypes  JenAge,GSE102434,Transcriptome Analysis,Comparison of gene expression profiles from Danio rerio skin of wiltype AB and homozygous/heterozygous tert mutant phenotypes AB tert /   AB tert+/. The RNA seq data comprises 3 groups. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: Overall Design: 13 samples in 3 groups: AB 5 samples  AB tert /  3 samples  AB tert+/  5 samples,,pubmed:34680427,,NH FLI 258 skin RNAseq,GSM2736509,,source name:skin|strain:AB tert+/ |tissue:skin|age:3 month|Sex:male,NH FLI 258 skin RNAseq,Sequence information was extracted in FastQ format using bcl2fastq software 1.8.4 data processing step. Reads were mapped using STAR 2.4.1d. Reads per gene were counted using featureCounts 1.4.6 p4. Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Genome build: genome: GRCz10  and respective gene annotation GRCz10.84 Supplementary files format and content: The Excel files include raw counts sample counts.xls and RPKM values sample rpkms.xls of all genes for each sample.,skin,,Extraction was done as described in Baumgart et al. 2012. PMID:22487494 Library preparation was done using Illumina's TruSeq RNA Library Preparation Kit v2 following the manufacturer's instruction.,,strain:AB tert+/ |tissue:skin|age:3 month|Sex:male,GSM2736509,GSM2736509: NH FLI 258 skin RNAseq; Danio rerio; RNA Seq,GSM2736509,,1,Extraction was done as described in Baumgart et al. 2012. PMID:22487494 Library preparation was done using Illumina's TruSeq RNA Library Preparation Kit v2 following the manufacturer's instruction.,GEO Accession:GSM2736509,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2500,,SRP115124,,,,,1205306664.0,23633464.0,GSM2736509 r1,0:51,A:343477875;C:261852731;G:256226343;T:343668156;N:81559,51,,,,343477875,261852731,256226343,343668156,81559,SRX3081853,SRS2420477,SRA597914,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.94347,,0.06609,,0.75513,,0.67529,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2017-08-09,Adult,Adult,Skin,Surface Structure
43252,SRR5921025,SRX3081852,SRS2420478,SRP115124,PRJNA397736,Sequencing of Danio rerio skin from wildtype and two tert mutant phenotypes  JenAge,GSE102434,Transcriptome Analysis,Comparison of gene expression profiles from Danio rerio skin of wiltype AB and homozygous/heterozygous tert mutant phenotypes AB tert /   AB tert+/. The RNA seq data comprises 3 groups. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: Overall Design: 13 samples in 3 groups: AB 5 samples  AB tert /  3 samples  AB tert+/  5 samples,,pubmed:34680427,,NH FLI 257 skin RNAseq,GSM2736508,,source name:skin|strain:AB wildtype|tissue:skin|age:3 month|Sex:male,NH FLI 257 skin RNAseq,Sequence information was extracted in FastQ format using bcl2fastq software 1.8.4 data processing step. Reads were mapped using STAR 2.4.1d. Reads per gene were counted using featureCounts 1.4.6 p4. Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Genome build: genome: GRCz10  and respective gene annotation GRCz10.84 Supplementary files format and content: The Excel files include raw counts sample counts.xls and RPKM values sample rpkms.xls of all genes for each sample.,skin,,Extraction was done as described in Baumgart et al. 2012. PMID:22487494 Library preparation was done using Illumina's TruSeq RNA Library Preparation Kit v2 following the manufacturer's instruction.,,strain:AB wildtype|tissue:skin|age:3 month|Sex:male,GSM2736508,GSM2736508: NH FLI 257 skin RNAseq; Danio rerio; RNA Seq,GSM2736508,,1,Extraction was done as described in Baumgart et al. 2012. PMID:22487494 Library preparation was done using Illumina's TruSeq RNA Library Preparation Kit v2 following the manufacturer's instruction.,GEO Accession:GSM2736508,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2500,,SRP115124,,,,,1149170556.0,22532756.0,GSM2736508 r1,0:51,A:322046038;C:255088738;G:249324620;T:322618409;N:92751,51,,,,322046038,255088738,249324620,322618409,92751,SRX3081852,SRS2420478,SRA597914,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.94133,,0.06843,,0.75917,,0.66656,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2017-08-09,Adult,Adult,Skin,Surface Structure
43253,SRR5921024,SRX3081851,SRS2420479,SRP115124,PRJNA397736,Sequencing of Danio rerio skin from wildtype and two tert mutant phenotypes  JenAge,GSE102434,Transcriptome Analysis,Comparison of gene expression profiles from Danio rerio skin of wiltype AB and homozygous/heterozygous tert mutant phenotypes AB tert /   AB tert+/. The RNA seq data comprises 3 groups. Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: Overall Design: 13 samples in 3 groups: AB 5 samples  AB tert /  3 samples  AB tert+/  5 samples,,pubmed:34680427,,NH FLI 256 skin RNAseq,GSM2736507,,source name:skin|strain:AB tert / |tissue:skin|age:3 month|Sex:male,NH FLI 256 skin RNAseq,Sequence information was extracted in FastQ format using bcl2fastq software 1.8.4 data processing step. Reads were mapped using STAR 2.4.1d. Reads per gene were counted using featureCounts 1.4.6 p4. Counts were normalized to RPKM values as described in Mortazavi et al. 2008. Genome build: genome: GRCz10  and respective gene annotation GRCz10.84 Supplementary files format and content: The Excel files include raw counts sample counts.xls and RPKM values sample rpkms.xls of all genes for each sample.,skin,,Extraction was done as described in Baumgart et al. 2012. PMID:22487494 Library preparation was done using Illumina's TruSeq RNA Library Preparation Kit v2 following the manufacturer's instruction.,,strain:AB tert / |tissue:skin|age:3 month|Sex:male,GSM2736507,GSM2736507: NH FLI 256 skin RNAseq; Danio rerio; RNA Seq,GSM2736507,,1,Extraction was done as described in Baumgart et al. 2012. PMID:22487494 Library preparation was done using Illumina's TruSeq RNA Library Preparation Kit v2 following the manufacturer's instruction.,GEO Accession:GSM2736507,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2500,,SRP115124,,,,,1626781476.0,31897676.0,GSM2736507 r1,0:51,A:455682785;C:362013305;G:353195709;T:455790202;N:99475,51,,,,455682785,362013305,353195709,455790202,99475,SRX3081851,SRS2420479,SRA597914,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.95384,,0.05948,,0.76648,,0.68751,,51,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,trueseq,bulk,unknown,unknown,,Germany,2017-08-09,Adult,Adult,Skin,Surface Structure