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
33892,SRR30866052,SRX26263961,SRS22803217,SRP536276,PRJNA1168147,Mgat4b mediated selective N glycosyl modification regulates melanocyte development and melanoma progression [scRNA seq],GSE278654,Transcriptome Analysis,Dysregulated melanocyte state transitions are a pivotal driver of melanoma development  highlighting the need to identify key regulators of these processes. Understanding these factors is key to know how normal melanocyte functions and shift towards initiation of melanoma. Our study identifies Mgat4b  a glycosyl transferase involved in selective N glycan branching enriched in pigment progenitors  as a key regulator of directional melanocyte migration and establishment of Melanocyte stem cell McSC pool during early development in zebrafish and mammalian melanocytes. Single cell RNA sequencing analysis in zebrafish upon targeted disruption of Mgat4b reveals that a subset of melanocytes marked by aberrant galectin expression are impaired in migration and are lost. Lectin binding proteomic analysis reveals the glycosylation of key melanocyte proteins Gpnmb  Kit  and Tyrp1 to be under the control of Mgat4b. Additionally  mislocalization of Gamma catenin Jup explains the observed defects in cell adhesion and migration to be regulated by mgat4b but not its isozyme mgat4a. Our meta analysis further revealed that melanoma patients with both the BrafV600E mutation and elevated Mgat4b levels have significantly worse survival outcomes compared to those with only the BrafV600E mutation. By leveraging the MAZERATI platform to model BrafV600E driver mutation in vivo  we show that Mgat4b mutant cells fail to aggregate and initiate tumors. Our study underscores the importance of selective N glycan branching in both melanocyte development and melanoma initiation  suggesting a Mitf controlled Mgat4b as a promising therapeutic target for melanoma treatment. Overall design: To understand how selective N glycosylation modification performed by mgat4b promote melanocyte development  we performed single cell RNA sequencing of mitfa+ve melanophores sorted from the 36hpf staged zebrafish.,,,,m4b mut,GSM8552316,,tissue:melanocytes|cell type:melanocytes|age:36 hpf|genotype:mgat4b mutant|treatment:No|geo loc name:missing|collection date:missing,m4b mut,Cellranger mkfastq pipeline was used to generate fastq files The fastq files were then aligned with custom zebrafish reference assembly GRCz11 using cellranger. Assembly: GRCz11 Supplementary files format and content: tab delimited text file including barcodes and features  and matrix files,melanocytes,,The gfp positive cells are isolated using FACS and 10x Genomics Next GEM three primereagent kit Dual Index was used for bead generation and cDNA synthesis 10x Genomics Next GEM three primereagent kit Dual Index was used for library construction,,cell type:melanocytes|age:36 hpf|genotype:mgat4b mutant|treatment:No,GSM8552316,GSM8552316: m4b mut; Danio rerio; RNA Seq,GSM8552316 r1,GSM8552316,1,The gfp positive cells are isolated using FACS and 10x Genomics Next GEM three primereagent kit Dual Index was used for bead generation and cDNA synthesis 10x Genomics Next GEM three primereagent kit Dual Index was used for library construction,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 2000,,SRP536276,,loader:fastq load.py,m4b_mut_S2_L001_I1_001.fastq.gz m4b_mut_S2_L001_I2_001.fastq.gz m4b_mut_S2_L001_R1_001.fastq.gz m4b_mut_S2_L001_R2_001.fastq.gz,fastq fastq fastq fastq,18274385040.0,132423080.0,GSM8552316 r1,0:10 1:10 2:28 3:90,A:3788703303;C:2355223955;G:2645521958;T:3126630009;N:1997975,10,10,28,90,3788703303,2355223955,2645521958,3126630009,1997975,SRX26263961,SRS22803217,SRA1984944,"Pigment Cell Biology Lab, CSIR-IGIB","Pigment Cell Biology Lab, CSIR-IGIB",,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq_v2,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,India,2024-10-02,Pharyngula,Embryo,Skin,Surface Structure
33893,SRR30866053,SRX26263960,SRS22803216,SRP536276,PRJNA1168147,Mgat4b mediated selective N glycosyl modification regulates melanocyte development and melanoma progression [scRNA seq],GSE278654,Transcriptome Analysis,Dysregulated melanocyte state transitions are a pivotal driver of melanoma development  highlighting the need to identify key regulators of these processes. Understanding these factors is key to know how normal melanocyte functions and shift towards initiation of melanoma. Our study identifies Mgat4b  a glycosyl transferase involved in selective N glycan branching enriched in pigment progenitors  as a key regulator of directional melanocyte migration and establishment of Melanocyte stem cell McSC pool during early development in zebrafish and mammalian melanocytes. Single cell RNA sequencing analysis in zebrafish upon targeted disruption of Mgat4b reveals that a subset of melanocytes marked by aberrant galectin expression are impaired in migration and are lost. Lectin binding proteomic analysis reveals the glycosylation of key melanocyte proteins Gpnmb  Kit  and Tyrp1 to be under the control of Mgat4b. Additionally  mislocalization of Gamma catenin Jup explains the observed defects in cell adhesion and migration to be regulated by mgat4b but not its isozyme mgat4a. Our meta analysis further revealed that melanoma patients with both the BrafV600E mutation and elevated Mgat4b levels have significantly worse survival outcomes compared to those with only the BrafV600E mutation. By leveraging the MAZERATI platform to model BrafV600E driver mutation in vivo  we show that Mgat4b mutant cells fail to aggregate and initiate tumors. Our study underscores the importance of selective N glycan branching in both melanocyte development and melanoma initiation  suggesting a Mitf controlled Mgat4b as a promising therapeutic target for melanoma treatment. Overall design: To understand how selective N glycosylation modification performed by mgat4b promote melanocyte development  we performed single cell RNA sequencing of mitfa+ve melanophores sorted from the 36hpf staged zebrafish.,,,,Control,GSM8552315,,tissue:melanocytes|cell type:melanocytes|age:36 hpf|genotype:control|treatment:No|geo loc name:missing|collection date:missing,Control,Cellranger mkfastq pipeline was used to generate fastq files The fastq files were then aligned with custom zebrafish reference assembly GRCz11 using cellranger. Assembly: GRCz11 Supplementary files format and content: tab delimited text file including barcodes and features  and matrix files,melanocytes,,The gfp positive cells are isolated using FACS and 10x Genomics Next GEM three primereagent kit Dual Index was used for bead generation and cDNA synthesis 10x Genomics Next GEM three primereagent kit Dual Index was used for library construction,,cell type:melanocytes|age:36 hpf|genotype:control|treatment:No,GSM8552315,GSM8552315: Control; Danio rerio; RNA Seq,GSM8552315 r1,GSM8552315,1,The gfp positive cells are isolated using FACS and 10x Genomics Next GEM three primereagent kit Dual Index was used for bead generation and cDNA synthesis 10x Genomics Next GEM three primereagent kit Dual Index was used for library construction,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 2000,,SRP536276,,loader:fastq load.py,con_S1_L001_I1_001.fastq.gz con_S1_L001_I2_001.fastq.gz con_S1_L001_R1_001.fastq.gz con_S1_L001_R2_001.fastq.gz,fastq fastq fastq fastq,15825218034.0,114675493.0,GSM8552315 r1,0:10 1:10 2:28 3:90,A:3074750870;C:2137365424;G:2401965854;T:2704984993;N:1727229,10,10,28,90,3074750870,2137365424,2401965854,2704984993,1727229,SRX26263960,SRS22803216,SRA1984944,"Pigment Cell Biology Lab, CSIR-IGIB","Pigment Cell Biology Lab, CSIR-IGIB",,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq_v2,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,India,2024-10-02,Pharyngula,Embryo,Skin,Surface Structure
33894,SRR30866028,SRX26263941,SRS22803195,SRP536275,PRJNA1168148,Mgat4b mediated selective N glycosyl modification regulates melanocyte development and melanoma progression [bulk RNA seq],GSE278653,Transcriptome Analysis,Dysregulated melanocyte state transitions are a pivotal driver of melanoma development  highlighting the need to identify key regulators of these processes. Understanding these factors is key to know how normal melanocyte functions and shift towards initiation of melanoma. Our study identifies Mgat4b  a glycosyl transferase involved in selective N glycan branching enriched in pigment progenitors  as a key regulator of directional melanocyte migration and establishment of Melanocyte stem cell McSC pool during early development in zebrafish and mammalian melanocytes. Single cell RNA sequencing analysis in zebrafish upon targeted disruption of Mgat4b reveals that a subset of melanocytes marked by aberrant galectin expression are impaired in migration and are lost. Lectin binding proteomic analysis reveals the glycosylation of key melanocyte proteins Gpnmb  Kit  and Tyrp1 to be under the control of Mgat4b. Additionally  mislocalization of Gamma catenin Jup explains the observed defects in cell adhesion and migration to be regulated by mgat4b but not its isozyme mgat4a. Our meta analysis further revealed that melanoma patients with both the BrafV600E mutation and elevated Mgat4b levels have significantly worse survival outcomes compared to those with only the BrafV600E mutation. By leveraging the MAZERATI platform to model BrafV600E driver mutation in vivo  we show that Mgat4b mutant cells fail to aggregate and initiate tumors. Our study underscores the importance of selective N glycan branching in both melanocyte development and melanoma initiation  suggesting a Mitf controlled Mgat4b as a promising therapeutic target for melanoma treatment. Overall design: To investigate the mechanisms that inhibit mutant cells from initiating melanoma in the absence of mgat4b  we utilized mature melanophores and melanoma cells from the skin of five zebrafish  including both MAZERATI wild type and  MAZERATI zebrafish carrying mgat4b mutations. We then performed gene expression profiling analysis using data obtained from RNA seq of melanophores and melanoma cells from both the zebrafish wild type and m4b mut. Comparative gene expression analysis was performed for wild type melanoma and melanophores  m4b mut melanoma and melanophores  wild type and m4b mut melanoma.,,,,m4b mut melanoma  biol rep 2,GSM8552314,,source name:skin|tissue:skin|cell type:melanoma|genotype:mgat4b mutant|treatment:MAZERATI|geo loc name:missing|collection date:missing,m4b mut melanoma  biol rep 2,Raw fastq files were trimmed using trimmomatic to remove adaptors and retain high quality reads. The reads were then aligned with zebrafish reference assembly GRCz11 using STAR. Featurecounts was used to calculate raw counts from the aligned reads. Assembly: GRCz11 Supplementary files format and content: tab delimited text file including raw counts for each sample,skin,,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,tissue:skin|cell type:melanoma|genotype:mgat4b mutant|treatment:MAZERATI,GSM8552314,GSM8552314: m4b mut melanoma  biol rep 2; Danio rerio; RNA Seq,GSM8552314 r1,GSM8552314,1,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 2000,,SRP536275,,,MKO2_S39_R1_001.fastq.gz MKO2_S39_R2_001.fastq.gz,fastq fastq,3705364625.0,14164066.0,GSM8552314 r1,0:126.72 1:134.89,A:785806700;C:949078797;G:1178279032;T:784972987;N:7227109,126,134,,,785806700,949078797,1178279032,784972987,7227109,SRX26263941,SRS22803195,SRA1984938,"Pigment Cell Biology Lab, CSIR-IGIB","Pigment Cell Biology Lab, CSIR-IGIB",,,,,,,,,,,,B,B,biological fallback assumption,illumina,nextseq_v2,unknown,cdna_unspecified,trueseq,sc_generic,bulk,bulk,,India,2024-10-02,Undetermined,Undetermined,Skin,Surface Structure
33895,SRR30866029,SRX26263940,SRS22803194,SRP536275,PRJNA1168148,Mgat4b mediated selective N glycosyl modification regulates melanocyte development and melanoma progression [bulk RNA seq],GSE278653,Transcriptome Analysis,Dysregulated melanocyte state transitions are a pivotal driver of melanoma development  highlighting the need to identify key regulators of these processes. Understanding these factors is key to know how normal melanocyte functions and shift towards initiation of melanoma. Our study identifies Mgat4b  a glycosyl transferase involved in selective N glycan branching enriched in pigment progenitors  as a key regulator of directional melanocyte migration and establishment of Melanocyte stem cell McSC pool during early development in zebrafish and mammalian melanocytes. Single cell RNA sequencing analysis in zebrafish upon targeted disruption of Mgat4b reveals that a subset of melanocytes marked by aberrant galectin expression are impaired in migration and are lost. Lectin binding proteomic analysis reveals the glycosylation of key melanocyte proteins Gpnmb  Kit  and Tyrp1 to be under the control of Mgat4b. Additionally  mislocalization of Gamma catenin Jup explains the observed defects in cell adhesion and migration to be regulated by mgat4b but not its isozyme mgat4a. Our meta analysis further revealed that melanoma patients with both the BrafV600E mutation and elevated Mgat4b levels have significantly worse survival outcomes compared to those with only the BrafV600E mutation. By leveraging the MAZERATI platform to model BrafV600E driver mutation in vivo  we show that Mgat4b mutant cells fail to aggregate and initiate tumors. Our study underscores the importance of selective N glycan branching in both melanocyte development and melanoma initiation  suggesting a Mitf controlled Mgat4b as a promising therapeutic target for melanoma treatment. Overall design: To investigate the mechanisms that inhibit mutant cells from initiating melanoma in the absence of mgat4b  we utilized mature melanophores and melanoma cells from the skin of five zebrafish  including both MAZERATI wild type and  MAZERATI zebrafish carrying mgat4b mutations. We then performed gene expression profiling analysis using data obtained from RNA seq of melanophores and melanoma cells from both the zebrafish wild type and m4b mut. Comparative gene expression analysis was performed for wild type melanoma and melanophores  m4b mut melanoma and melanophores  wild type and m4b mut melanoma.,,,,m4b mut melanoma  biol rep 1,GSM8552313,,source name:skin|tissue:skin|cell type:melanoma|genotype:mgat4b mutant|treatment:MAZERATI|geo loc name:missing|collection date:missing,m4b mut melanoma  biol rep 1,Raw fastq files were trimmed using trimmomatic to remove adaptors and retain high quality reads. The reads were then aligned with zebrafish reference assembly GRCz11 using STAR. Featurecounts was used to calculate raw counts from the aligned reads. Assembly: GRCz11 Supplementary files format and content: tab delimited text file including raw counts for each sample,skin,,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,tissue:skin|cell type:melanoma|genotype:mgat4b mutant|treatment:MAZERATI,GSM8552313,GSM8552313: m4b mut melanoma  biol rep 1; Danio rerio; RNA Seq,GSM8552313 r1,GSM8552313,1,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 2000,,SRP536275,,,MKO1_S38_R1_001.fastq.gz MKO1_S38_R2_001.fastq.gz,fastq fastq,4779377372.0,18058294.0,GSM8552313 r1,0:128.72 1:135.94,A:1016908920;C:1236613888;G:1502465334;T:1018053808;N:5335422,128,135,,,1016908920,1236613888,1502465334,1018053808,5335422,SRX26263940,SRS22803194,SRA1984938,"Pigment Cell Biology Lab, CSIR-IGIB","Pigment Cell Biology Lab, CSIR-IGIB",,,,,,,,,,,,B,B,biological fallback assumption,illumina,nextseq_v2,unknown,cdna_unspecified,trueseq,sc_generic,bulk,bulk,,India,2024-10-02,Undetermined,Undetermined,Skin,Surface Structure
33896,SRR30866030,SRX26263939,SRS22803193,SRP536275,PRJNA1168148,Mgat4b mediated selective N glycosyl modification regulates melanocyte development and melanoma progression [bulk RNA seq],GSE278653,Transcriptome Analysis,Dysregulated melanocyte state transitions are a pivotal driver of melanoma development  highlighting the need to identify key regulators of these processes. Understanding these factors is key to know how normal melanocyte functions and shift towards initiation of melanoma. Our study identifies Mgat4b  a glycosyl transferase involved in selective N glycan branching enriched in pigment progenitors  as a key regulator of directional melanocyte migration and establishment of Melanocyte stem cell McSC pool during early development in zebrafish and mammalian melanocytes. Single cell RNA sequencing analysis in zebrafish upon targeted disruption of Mgat4b reveals that a subset of melanocytes marked by aberrant galectin expression are impaired in migration and are lost. Lectin binding proteomic analysis reveals the glycosylation of key melanocyte proteins Gpnmb  Kit  and Tyrp1 to be under the control of Mgat4b. Additionally  mislocalization of Gamma catenin Jup explains the observed defects in cell adhesion and migration to be regulated by mgat4b but not its isozyme mgat4a. Our meta analysis further revealed that melanoma patients with both the BrafV600E mutation and elevated Mgat4b levels have significantly worse survival outcomes compared to those with only the BrafV600E mutation. By leveraging the MAZERATI platform to model BrafV600E driver mutation in vivo  we show that Mgat4b mutant cells fail to aggregate and initiate tumors. Our study underscores the importance of selective N glycan branching in both melanocyte development and melanoma initiation  suggesting a Mitf controlled Mgat4b as a promising therapeutic target for melanoma treatment. Overall design: To investigate the mechanisms that inhibit mutant cells from initiating melanoma in the absence of mgat4b  we utilized mature melanophores and melanoma cells from the skin of five zebrafish  including both MAZERATI wild type and  MAZERATI zebrafish carrying mgat4b mutations. We then performed gene expression profiling analysis using data obtained from RNA seq of melanophores and melanoma cells from both the zebrafish wild type and m4b mut. Comparative gene expression analysis was performed for wild type melanoma and melanophores  m4b mut melanoma and melanophores  wild type and m4b mut melanoma.,,,,wild type melanoma control  biol rep 2,GSM8552312,,source name:skin|tissue:skin|cell type:melanoma|genotype:WT|treatment:MAZERATI|geo loc name:missing|collection date:missing,wild type melanoma control  biol rep 2,Raw fastq files were trimmed using trimmomatic to remove adaptors and retain high quality reads. The reads were then aligned with zebrafish reference assembly GRCz11 using STAR. Featurecounts was used to calculate raw counts from the aligned reads. Assembly: GRCz11 Supplementary files format and content: tab delimited text file including raw counts for each sample,skin,,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,tissue:skin|cell type:melanoma|genotype:WT|treatment:MAZERATI,GSM8552312,GSM8552312: wild type melanoma control  biol rep 2; Danio rerio; RNA Seq,GSM8552312 r1,GSM8552312,1,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 2000,,SRP536275,,,EV2_S37_R1_001.fastq.gz EV2_S37_R2_001.fastq.gz,fastq fastq,5427379509.0,19981504.0,GSM8552312 r1,0:133.16 1:138.46,A:1136639418;C:1414638483;G:1736492639;T:1137614638;N:1994331,133,138,,,1136639418,1414638483,1736492639,1137614638,1994331,SRX26263939,SRS22803193,SRA1984938,"Pigment Cell Biology Lab, CSIR-IGIB","Pigment Cell Biology Lab, CSIR-IGIB",,,,,,,,,,,,B,B,biological fallback assumption,illumina,nextseq_v2,unknown,cdna_unspecified,trueseq,sc_generic,bulk,bulk,,India,2024-10-02,Undetermined,Undetermined,Skin,Surface Structure
33897,SRR30866031,SRX26263938,SRS22803192,SRP536275,PRJNA1168148,Mgat4b mediated selective N glycosyl modification regulates melanocyte development and melanoma progression [bulk RNA seq],GSE278653,Transcriptome Analysis,Dysregulated melanocyte state transitions are a pivotal driver of melanoma development  highlighting the need to identify key regulators of these processes. Understanding these factors is key to know how normal melanocyte functions and shift towards initiation of melanoma. Our study identifies Mgat4b  a glycosyl transferase involved in selective N glycan branching enriched in pigment progenitors  as a key regulator of directional melanocyte migration and establishment of Melanocyte stem cell McSC pool during early development in zebrafish and mammalian melanocytes. Single cell RNA sequencing analysis in zebrafish upon targeted disruption of Mgat4b reveals that a subset of melanocytes marked by aberrant galectin expression are impaired in migration and are lost. Lectin binding proteomic analysis reveals the glycosylation of key melanocyte proteins Gpnmb  Kit  and Tyrp1 to be under the control of Mgat4b. Additionally  mislocalization of Gamma catenin Jup explains the observed defects in cell adhesion and migration to be regulated by mgat4b but not its isozyme mgat4a. Our meta analysis further revealed that melanoma patients with both the BrafV600E mutation and elevated Mgat4b levels have significantly worse survival outcomes compared to those with only the BrafV600E mutation. By leveraging the MAZERATI platform to model BrafV600E driver mutation in vivo  we show that Mgat4b mutant cells fail to aggregate and initiate tumors. Our study underscores the importance of selective N glycan branching in both melanocyte development and melanoma initiation  suggesting a Mitf controlled Mgat4b as a promising therapeutic target for melanoma treatment. Overall design: To investigate the mechanisms that inhibit mutant cells from initiating melanoma in the absence of mgat4b  we utilized mature melanophores and melanoma cells from the skin of five zebrafish  including both MAZERATI wild type and  MAZERATI zebrafish carrying mgat4b mutations. We then performed gene expression profiling analysis using data obtained from RNA seq of melanophores and melanoma cells from both the zebrafish wild type and m4b mut. Comparative gene expression analysis was performed for wild type melanoma and melanophores  m4b mut melanoma and melanophores  wild type and m4b mut melanoma.,,,,wild type melanoma control  biol rep 1,GSM8552311,,source name:skin|tissue:skin|cell type:melanoma|genotype:WT|treatment:MAZERATI|geo loc name:missing|collection date:missing,wild type melanoma control  biol rep 1,Raw fastq files were trimmed using trimmomatic to remove adaptors and retain high quality reads. The reads were then aligned with zebrafish reference assembly GRCz11 using STAR. Featurecounts was used to calculate raw counts from the aligned reads. Assembly: GRCz11 Supplementary files format and content: tab delimited text file including raw counts for each sample,skin,,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,tissue:skin|cell type:melanoma|genotype:WT|treatment:MAZERATI,GSM8552311,GSM8552311: wild type melanoma control  biol rep 1; Danio rerio; RNA Seq,GSM8552311 r1,GSM8552311,1,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 2000,,SRP536275,,,EV1_S36_R1_001.fastq.gz EV1_S36_R2_001.fastq.gz,fastq fastq,5248108957.0,19087024.0,GSM8552311 r1,0:135.12 1:139.84,A:1112163842;C:1354895660;G:1658732932;T:1120342985;N:1973538,135,139,,,1112163842,1354895660,1658732932,1120342985,1973538,SRX26263938,SRS22803192,SRA1984938,"Pigment Cell Biology Lab, CSIR-IGIB","Pigment Cell Biology Lab, CSIR-IGIB",,,,,,,,,,,,B,B,biological fallback assumption,illumina,nextseq_v2,unknown,cdna_unspecified,trueseq,sc_generic,bulk,bulk,,India,2024-10-02,Undetermined,Undetermined,Skin,Surface Structure
33898,SRR30866032,SRX26263937,SRS22803191,SRP536275,PRJNA1168148,Mgat4b mediated selective N glycosyl modification regulates melanocyte development and melanoma progression [bulk RNA seq],GSE278653,Transcriptome Analysis,Dysregulated melanocyte state transitions are a pivotal driver of melanoma development  highlighting the need to identify key regulators of these processes. Understanding these factors is key to know how normal melanocyte functions and shift towards initiation of melanoma. Our study identifies Mgat4b  a glycosyl transferase involved in selective N glycan branching enriched in pigment progenitors  as a key regulator of directional melanocyte migration and establishment of Melanocyte stem cell McSC pool during early development in zebrafish and mammalian melanocytes. Single cell RNA sequencing analysis in zebrafish upon targeted disruption of Mgat4b reveals that a subset of melanocytes marked by aberrant galectin expression are impaired in migration and are lost. Lectin binding proteomic analysis reveals the glycosylation of key melanocyte proteins Gpnmb  Kit  and Tyrp1 to be under the control of Mgat4b. Additionally  mislocalization of Gamma catenin Jup explains the observed defects in cell adhesion and migration to be regulated by mgat4b but not its isozyme mgat4a. Our meta analysis further revealed that melanoma patients with both the BrafV600E mutation and elevated Mgat4b levels have significantly worse survival outcomes compared to those with only the BrafV600E mutation. By leveraging the MAZERATI platform to model BrafV600E driver mutation in vivo  we show that Mgat4b mutant cells fail to aggregate and initiate tumors. Our study underscores the importance of selective N glycan branching in both melanocyte development and melanoma initiation  suggesting a Mitf controlled Mgat4b as a promising therapeutic target for melanoma treatment. Overall design: To investigate the mechanisms that inhibit mutant cells from initiating melanoma in the absence of mgat4b  we utilized mature melanophores and melanoma cells from the skin of five zebrafish  including both MAZERATI wild type and  MAZERATI zebrafish carrying mgat4b mutations. We then performed gene expression profiling analysis using data obtained from RNA seq of melanophores and melanoma cells from both the zebrafish wild type and m4b mut. Comparative gene expression analysis was performed for wild type melanoma and melanophores  m4b mut melanoma and melanophores  wild type and m4b mut melanoma.,,,,melanophore  biol rep 2,GSM8552310,,source name:skin|tissue:skin|cell type:melanophore|genotype:WT|treatment:No|geo loc name:missing|collection date:missing,melanophore  biol rep 2,Raw fastq files were trimmed using trimmomatic to remove adaptors and retain high quality reads. The reads were then aligned with zebrafish reference assembly GRCz11 using STAR. Featurecounts was used to calculate raw counts from the aligned reads. Assembly: GRCz11 Supplementary files format and content: tab delimited text file including raw counts for each sample,skin,,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,tissue:skin|cell type:melanophore|genotype:WT|treatment:No,GSM8552310,GSM8552310: melanophore  biol rep 2; Danio rerio; RNA Seq,GSM8552310 r1,GSM8552310,1,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 2000,,SRP536275,,,WT2_S41_R1_001.fastq.gz WT2_S41_R2_001.fastq.gz,fastq fastq,4636631881.0,16340506.0,GSM8552310 r1,0:140.47 1:143.28,A:1114533149;C:1104066636;G:1293649625;T:1122672029;N:1710442,140,143,,,1114533149,1104066636,1293649625,1122672029,1710442,SRX26263937,SRS22803191,SRA1984938,"Pigment Cell Biology Lab, CSIR-IGIB","Pigment Cell Biology Lab, CSIR-IGIB",,,,,,,,,,,,B,B,biological fallback assumption,illumina,nextseq_v2,unknown,cdna_unspecified,trueseq,sc_generic,bulk,bulk,,India,2024-10-02,Undetermined,Undetermined,Skin,Surface Structure
33899,SRR30866033,SRX26263936,SRS22803190,SRP536275,PRJNA1168148,Mgat4b mediated selective N glycosyl modification regulates melanocyte development and melanoma progression [bulk RNA seq],GSE278653,Transcriptome Analysis,Dysregulated melanocyte state transitions are a pivotal driver of melanoma development  highlighting the need to identify key regulators of these processes. Understanding these factors is key to know how normal melanocyte functions and shift towards initiation of melanoma. Our study identifies Mgat4b  a glycosyl transferase involved in selective N glycan branching enriched in pigment progenitors  as a key regulator of directional melanocyte migration and establishment of Melanocyte stem cell McSC pool during early development in zebrafish and mammalian melanocytes. Single cell RNA sequencing analysis in zebrafish upon targeted disruption of Mgat4b reveals that a subset of melanocytes marked by aberrant galectin expression are impaired in migration and are lost. Lectin binding proteomic analysis reveals the glycosylation of key melanocyte proteins Gpnmb  Kit  and Tyrp1 to be under the control of Mgat4b. Additionally  mislocalization of Gamma catenin Jup explains the observed defects in cell adhesion and migration to be regulated by mgat4b but not its isozyme mgat4a. Our meta analysis further revealed that melanoma patients with both the BrafV600E mutation and elevated Mgat4b levels have significantly worse survival outcomes compared to those with only the BrafV600E mutation. By leveraging the MAZERATI platform to model BrafV600E driver mutation in vivo  we show that Mgat4b mutant cells fail to aggregate and initiate tumors. Our study underscores the importance of selective N glycan branching in both melanocyte development and melanoma initiation  suggesting a Mitf controlled Mgat4b as a promising therapeutic target for melanoma treatment. Overall design: To investigate the mechanisms that inhibit mutant cells from initiating melanoma in the absence of mgat4b  we utilized mature melanophores and melanoma cells from the skin of five zebrafish  including both MAZERATI wild type and  MAZERATI zebrafish carrying mgat4b mutations. We then performed gene expression profiling analysis using data obtained from RNA seq of melanophores and melanoma cells from both the zebrafish wild type and m4b mut. Comparative gene expression analysis was performed for wild type melanoma and melanophores  m4b mut melanoma and melanophores  wild type and m4b mut melanoma.,,,,melanophore  biol rep 1,GSM8552309,,source name:skin|tissue:skin|cell type:melanophore|genotype:WT|treatment:No|geo loc name:missing|collection date:missing,melanophore  biol rep 1,Raw fastq files were trimmed using trimmomatic to remove adaptors and retain high quality reads. The reads were then aligned with zebrafish reference assembly GRCz11 using STAR. Featurecounts was used to calculate raw counts from the aligned reads. Assembly: GRCz11 Supplementary files format and content: tab delimited text file including raw counts for each sample,skin,,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,tissue:skin|cell type:melanophore|genotype:WT|treatment:No,GSM8552309,GSM8552309: melanophore  biol rep 1; Danio rerio; RNA Seq,GSM8552309 r1,GSM8552309,1,The cell isolation process involved density gradient centrifugation  followed by RNA extraction using Trizol method. Illumina standard total RNA prep kit was used for library construction,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 2000,,SRP536275,,,WT1_S40_R1_001.fastq.gz WT1_S40_R2_001.fastq.gz,fastq fastq,5185761246.0,18118534.0,GSM8552309 r1,0:141.81 1:144.40,A:1231235669;C:1235759770;G:1476864583;T:1240636871;N:1264353,141,144,,,1231235669,1235759770,1476864583,1240636871,1264353,SRX26263936,SRS22803190,SRA1984938,"Pigment Cell Biology Lab, CSIR-IGIB","Pigment Cell Biology Lab, CSIR-IGIB",,,,,,,,,,,,B,B,biological fallback assumption,illumina,nextseq_v2,unknown,cdna_unspecified,trueseq,sc_generic,bulk,bulk,,India,2024-10-02,Undetermined,Undetermined,Skin,Surface Structure
36385,SRR516135,SRX156099,SRS346038,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB  5m  skin  mRNA,zebrafish Nr31 5m,,,,,,,,,,,1 male Danio rerio strain TueAB  5m  skin  mRNA  Illumina GA IIx  76 bp,zebrafish Nr31 5m 1,zebrafish Nr31 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013931,,,,,1482677008.0,19508908.0,zebrafish Nr31 5m 1 1,0:76,A:385413939;C:354317020;G:354043481;T:388361606;N:540962,76,,,,385413939,354317020,354043481,388361606,540962,SRX156099,SRS346038,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.9273,,0.08335,,0.67898,,0.48956,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36386,SRR516140,SRX156099,SRS346038,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB  5m  skin  mRNA,zebrafish Nr31 5m,,,,,,,,,,,1 male Danio rerio strain TueAB  5m  skin  mRNA  Illumina GA IIx  76 bp,zebrafish Nr31 5m 1,zebrafish Nr31 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013931,,,,,388560032.0,5112632.0,zebrafish Nr31 5m 1 2,0:76,A:102207965;C:91473514;G:92119606;T:102551859;N:207088,76,,,,102207965,91473514,92119606,102551859,207088,SRX156099,SRS346038,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92486,,0.08531,,0.67663,,0.49425,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36387,SRR516134,SRX156098,SRS346037,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB  5m  skin  mRNA,zebrafish Nr30 5m,,,,,,,,,,,1 male Danio rerio strain TueAB  5m  skin  mRNA  Illumina GA IIx  76 bp,zebrafish Nr30 5m 1,zebrafish Nr30 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013931,,,,,1675582184.0,22047134.0,zebrafish Nr30 5m 1 1,0:76,A:458929887;C:381230210;G:376734439;T:457916852;N:770796,76,,,,458929887,381230210,376734439,457916852,770796,SRX156098,SRS346037,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91832,,0.10303,,0.71455,,0.49444,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36388,SRR516139,SRX156098,SRS346037,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB  5m  skin  mRNA,zebrafish Nr30 5m,,,,,,,,,,,1 male Danio rerio strain TueAB  5m  skin  mRNA  Illumina GA IIx  76 bp,zebrafish Nr30 5m 1,zebrafish Nr30 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013931,,,,,477065908.0,6277183.0,zebrafish Nr30 5m 1 2,0:76,A:131927852;C:107181234;G:106214626;T:131489254;N:252942,76,,,,131927852,107181234,106214626,131489254,252942,SRX156098,SRS346037,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91581,,0.10754,,0.71577,,0.49994,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36389,SRR516133,SRX156097,SRS346036,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB  5m  skin  mRNA,zebrafish Nr29 5m,,,,,,,,,,,1 male Danio rerio strain TueAB  5m  skin  mRNA  Illumina GA IIx  76 bp,zebrafish Nr29 5m 1,zebrafish Nr29 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013931,,,,,1305047832.0,17171682.0,zebrafish Nr29 5m 1 1,0:76,A:362467093;C:292043132;G:289641844;T:360441501;N:454262,76,,,,362467093,292043132,289641844,360441501,454262,SRX156097,SRS346036,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.90448,,0.11905,,0.70246,,0.47656,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36390,SRR516138,SRX156097,SRS346036,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB  5m  skin  mRNA,zebrafish Nr29 5m,,,,,,,,,,,1 male Danio rerio strain TueAB  5m  skin  mRNA  Illumina GA IIx  76 bp,zebrafish Nr29 5m 1,zebrafish Nr29 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013931,,,110307_DanioRerio_Nr29_5m_w1_s_1.fq,fastq,457287972.0,6016947.0,zebrafish Nr29 5m 1 2,0:76,A:128212070;C:101000378;G:100136000;T:127695086;N:244438,76,,,,128212070,101000378,100136000,127695086,244438,SRX156097,SRS346036,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.90327,,0.12466,,0.70195,,0.49568,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2013-03-11,Adult,Adult,Skin,Surface Structure
36391,SRR516132,SRX156096,SRS346035,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB  5m  skin  mRNA,zebrafish Nr28 5m,,,,,,,,,,,1 male Danio rerio strain TueAB  5m  skin  mRNA  Illumina GA IIx  76 bp,zebrafish Nr28 5m 1,zebrafish Nr28 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013931,,,110104_DanioRerio_Nr28_5m_l5.fq,fastq,778479248.0,10243148.0,zebrafish Nr28 5m 1 1,0:76,A:207103962;C:183338330;G:183353815;T:204420105;N:263036,76,,,,207103962,183338330,183353815,204420105,263036,SRX156096,SRS346035,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91711,,0.08403,,0.72218,,0.46568,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2013-03-11,Adult,Adult,Skin,Surface Structure
36392,SRR516137,SRX156096,SRS346035,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB  5m  skin  mRNA,zebrafish Nr28 5m,,,,,,,,,,,1 male Danio rerio strain TueAB  5m  skin  mRNA  Illumina GA IIx  76 bp,zebrafish Nr28 5m 1,zebrafish Nr28 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013931,,,110307_DanioRerio_Nr28_5m_w1_s_2.fq,fastq,1004119144.0,13212094.0,zebrafish Nr28 5m 1 2,0:76,A:269579079;C:233472158;G:233160786;T:267709479;N:197642,76,,,,269579079,233472158,233160786,267709479,197642,SRX156096,SRS346035,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92125,,0.09157,,0.72056,,0.4782,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2013-03-11,Adult,Adult,Skin,Surface Structure
36393,SRR516131,SRX156095,SRS346034,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB  5m  skin  mRNA,zebrafish Nr27 5m,,,,,,,,,,,1 male Danio rerio strain TueAB  5m  skin  mRNA  Illumina GA IIx  76 bp,zebrafish Nr27 5m 1,zebrafish Nr27 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013931,,,,,1689987528.0,22236678.0,zebrafish Nr27 5m 1 1,0:76,A:462852366;C:385867681;G:380635625;T:460044585;N:587271,76,,,,462852366,385867681,380635625,460044585,587271,SRX156095,SRS346034,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91288,,0.1015,,0.71293,,0.488,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36394,SRR516136,SRX156095,SRS346034,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB  5m  skin  mRNA,zebrafish Nr27 5m,,,,,,,,,,,1 male Danio rerio strain TueAB  5m  skin  mRNA  Illumina GA IIx  76 bp,zebrafish Nr27 5m 1,zebrafish Nr27 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013931,,,,,421535824.0,5546524.0,zebrafish Nr27 5m 1 2,0:76,A:116434305;C:94958671;G:93876467;T:116039545;N:226836,76,,,,116434305,94958671,93876467,116039545,226836,SRX156095,SRS346034,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.90776,,0.10459,,0.71399,,0.49081,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36395,SRR516130,SRX156094,SRS346033,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB  5m  skin  mRNA,zebrafish Nr26 5m,,,,,,,,,,,1 male Danio rerio strain TueAB  5m  skin  mRNA  Illumina GA IIx  76 bp,zebrafish Nr26 5m 1,zebrafish Nr26 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013931,,,,,1633478944.0,21493144.0,zebrafish Nr26 5m 1 1,0:76,A:435251153;C:384938751;G:380973255;T:432171855;N:143930,76,,,,435251153,384938751,380973255,432171855,143930,SRX156094,SRS346033,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92486,,0.08081,,0.71969,,0.50656,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36396,SRR516129,SRX156093,SRS346032,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB  5m  skin  mRNA,zebrafish Nr25 5m,,,,,,,,,,,1 male Danio rerio strain TueAB  5m  skin  mRNA  Illumina GA IIx  76 bp,zebrafish Nr25 5m 1,zebrafish Nr25 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013931,,,101021_DanioRerio_Nr25_5m_l7.fq,fastq,1893537644.0,24914969.0,zebrafish Nr25 5m 1 1,0:76,A:516246577;C:431589332;G:431233057;T:514385629;N:83049,76,,,,516246577,431589332,431233057,514385629,83049,SRX156093,SRS346032,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.89513,,0.11188,,0.70591,,0.47827,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36397,SRR516128,SRX156092,SRS346031,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB  5m  skin  mRNA,zebrafish Nr23 5m,,,,,,,,,,,1 male Danio rerio strain TueAB  5m  skin  mRNA  Illumina GA IIx  76 bp,zebrafish Nr23 5m 1,zebrafish Nr23 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013931,,,101021_DanioRerio_Nr23_5m_l6.fq,fastq,2271040360.0,29882110.0,zebrafish Nr23 5m 1 1,0:76,A:602084142;C:529733757;G:524301688;T:614827528;N:93245,76,,,,602084142,529733757,524301688,614827528,93245,SRX156092,SRS346031,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92194,,0.09655,,0.70463,,0.47059,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2013-03-11,Adult,Adult,Skin,Surface Structure
36398,SRR516127,SRX156091,SRS346030,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB  5m  skin  mRNA,zebrafish Nr22 5m,,,,,,,,,,,1 male Danio rerio strain TueAB  5m  skin  mRNA  Illumina GA IIx  76 bp,zebrafish Nr22 5m 1,zebrafish Nr22 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013931,,,,,2812847324.0,37011149.0,zebrafish Nr22 5m 1 1,0:76,A:760320146;C:645617435;G:643672544;T:763112485;N:124714,76,,,,760320146,645617435,643672544,763112485,124714,SRX156091,SRS346030,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93272,,0.08997,,0.70755,,0.52272,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36399,SRR516126,SRX156090,SRS346029,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 5 month. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB  5m  skin  mRNA,zebrafish Nr21 5m,,,,,,,,,,,1 male Danio rerio strain TueAB  5m  skin  mRNA  Illumina GA IIx  76 bp,zebrafish Nr21 5m 1,zebrafish Nr21 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013931,,,,,2331404728.0,30676378.0,zebrafish Nr21 5m 1 1,0:76,A:630276643;C:539444103;G:529126097;T:632442560;N:115325,76,,,,630276643,539444103,529126097,632442560,115325,SRX156090,SRS346029,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92747,,0.10341,,0.70414,,0.49626,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36400,SRR516125,SRX156089,SRS346028,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 3.5 years. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB  3.5y  skin  mRNA,zebrafish Nr18 3.5y,,,,,,,,,,,1 male Danio rerio strain TueAB  3.5y  skin  mRNA  Illumina GA IIx  76 bp,zebrafish Nr18 3.5y 1,zebrafish Nr18 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013931,,,,,2653618888.0,34916038.0,zebrafish Nr18 3.5y 1 1,0:76,A:728660211;C:598216653;G:601771842;T:724871715;N:98467,76,,,,728660211,598216653,601771842,724871715,98467,SRX156089,SRS346028,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91227,,0.10895,,0.71802,,0.49946,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36401,SRR516124,SRX156088,SRS346027,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 3.5 years. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB  3.5y  skin  mRNA,zebrafish Nr14 3.5y,,,,,,,,,,,1 male Danio rerio strain TueAB  3.5y  skin  mRNA  Illumina GA IIx  76 bp,zebrafish Nr14 3.5y 1,zebrafish Nr14 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013931,,,,,2013201012.0,26489487.0,zebrafish Nr14 3.5y 1 1,0:76,A:554778161;C:456082219;G:445029202;T:557232699;N:78731,76,,,,554778161,456082219,445029202,557232699,78731,SRX156088,SRS346027,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.90845,,0.1164,,0.71236,,0.48939,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36402,SRR516123,SRX156087,SRS346026,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 3.5 years. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB  3.5y  skin  mRNA,zebrafish Nr13 3.5y,,,,,,,,,,,1 male Danio rerio strain TueAB  3.5y  skin  mRNA  Illumina GA IIx  76 bp,zebrafish Nr13 3.5y 1,zebrafish Nr13 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013931,,,,,2680273532.0,35266757.0,zebrafish Nr13 3.5y 1 1,0:76,A:740111520;C:588329849;G:606909161;T:744802954;N:120048,76,,,,740111520,588329849,606909161,744802954,120048,SRX156087,SRS346026,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.85899,,0.11949,,0.71595,,0.48655,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36403,SRR516122,SRX156086,SRS346025,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 3.5 years. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB  3.5y  skin  mRNA,zebrafish Nr12 3.5y,,,,,,,,,,,1 male Danio rerio strain TueAB  3.5y  skin  mRNA  Illumina GA IIx  76 bp,zebrafish Nr12 3.5y 1,zebrafish Nr12 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013931,,,,,2982635200.0,39245200.0,zebrafish Nr12 3.5y 1 1,0:76,A:829678392;C:650275344;G:659157904;T:842323646;N:1199914,76,,,,829678392,650275344,659157904,842323646,1199914,SRX156086,SRS346025,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.88932,,0.12521,,0.71638,,0.53514,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36404,SRR516121,SRX156085,SRS346024,SRP013931,PRJNA169239,Initial analysis of transcript levels in zebrafish with advancing age,PRJNA169239,Other,This project aims at an initial characterization of changes in gene expression in zebrafish with advancing age. Transcript levels are determined in several tissues of zebrafish with differing ages using RNA seq. Differentially expressed genes are determined to pinpoint genes that are differently regulated in young and old zebrafish. Results will be compared with other species to identify common pathways of ageing.,,,Wild type zebrafish of the TueAB strain were kept in groups and only males were used for RNA isolation at an age of 3.5 years. Total RNA was isolated from skin using Trizol Invitrogen.,1 male Danio rerio strain TueAB  3.5y  skin  mRNA,zebrafish Nr11 3.5y,,,,,,,,,,,1 male Danio rerio strain TueAB  3.5y  skin  mRNA  Illumina GA IIx  76 bp,zebrafish Nr11 3.5y 1,zebrafish Nr11 1,1,,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>76</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,SRP013931,,,,,2696915708.0,35485733.0,zebrafish Nr11 3.5y 1 1,0:76,A:749305835;C:599264137;G:607414252;T:740194221;N:737263,76,,,,749305835,599264137,607414252,740194221,737263,SRX156085,SRS346024,SRA054207,Leibniz Institute for Age Research - Fritz Lipmann|Genome Analysis,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.86902,,0.11557,,0.71461,,0.49255,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,Germany,2012-06-26,Adult,Adult,Skin,Surface Structure
36712,SRR835167,SRX271963,SRS416259,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 116h 53,GSM1129625,,tissue:melanocytes|hpf,dm 116h 53,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129625,GSM1129625: dm 116h 53; Danio rerio; RNA Seq,GSM1129625 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129625,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_116h_53_440.fq.bz2,fastq,203842506.0,4853393.0,GSM1129625 r1,0:42,A:49784918;C:48871504;G:45835011;T:59338271;N:12802,42,,,,49784918,48871504,45835011,59338271,12802,SRX271963,SRS416259,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.82741,,0.14973,,0.84893,,0.53644,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36713,SRR835166,SRX271962,SRS416258,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 97h 41 440,GSM1129624,,tissue:melanocytes|hpf,dm 97h 41 440,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129624,GSM1129624: dm 97h 41 440; Danio rerio; RNA Seq,GSM1129624 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129624,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_97h_41_440.fq.bz2,fastq,199638768.0,4753304.0,GSM1129624 r1,0:42,A:45958194;C:50367228;G:48451580;T:54849238;N:12528,42,,,,45958194,50367228,48451580,54849238,12528,SRX271962,SRS416258,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.85741,,0.09897,,0.84112,,0.53299,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36714,SRR835165,SRX271961,SRS416257,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h FM 351,GSM1129623,,tissue:melanocytes|hpf,dm 77h FM 351,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129623,GSM1129623: dm 77h FM 351; Danio rerio; RNA Seq,GSM1129623 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129623,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_FM_351_ATT.fq.bz2,fastq,194385384.0,5399594.0,GSM1129623 r1,0:36,A:45805148;C:47931006;G:41165284;T:59482433;N:1513,36,,,,45805148,47931006,41165284,59482433,1513,SRX271961,SRS416257,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.77748,,0.07764,,0.87687,,0.51575,,36,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36715,SRR835164,SRX271960,SRS416256,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 52 440,GSM1129622,,tissue:melanocytes|hpf,dm 77h 52 440,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129622,GSM1129622: dm 77h 52 440; Danio rerio; RNA Seq,GSM1129622 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129622,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_52_440.fq.bz2,fastq,105047586.0,2501133.0,GSM1129622 r1,0:42,A:24711000;C:26459041;G:24869198;T:29001957;N:6390,42,,,,24711000,26459041,24869198,29001957,6390,SRX271960,SRS416256,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.88862,,0.09219,,0.83611,,0.55476,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36716,SRR835163,SRX271959,SRS416255,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 43 440,GSM1129621,,tissue:melanocytes|hpf,dm 77h 43 440,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129621,GSM1129621: dm 77h 43 440; Danio rerio; RNA Seq,GSM1129621 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129621,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_43_440.fq.bz2,fastq,371553084.0,8846502.0,GSM1129621 r1,0:42,A:84994407;C:91269417;G:87188701;T:108077735;N:22824,42,,,,84994407,91269417,87188701,108077735,22824,SRX271959,SRS416255,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.86495,,0.09748,,0.83684,,0.52833,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36717,SRR835162,SRX271958,SRS416254,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 28 399s62,GSM1129620,,tissue:melanocytes|hpf,dm 77h 28 399s62,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129620,GSM1129620: dm 77h 28 399s62; Danio rerio; RNA Seq,GSM1129620 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129620,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,,,731276158.0,3620179.0,GSM1129620 r1,0:101 1:101,A:173989639;C:199270484;G:188281318;T:169725601;N:9116,101,101,,,173989639,199270484,188281318,169725601,9116,SRX271958,SRS416254,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",2,0.84122,0.79964,0.02274,0.02168,0.89645,0.89073,0.63341,0.63015,101,101,B,B,biological fallback assumption,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36718,SRR835161,SRX271957,SRS416253,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 28 399s61,GSM1129619,,tissue:melanocytes|hpf,dm 77h 28 399s61,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129619,GSM1129619: dm 77h 28 399s61; Danio rerio; RNA Seq,GSM1129619 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129619,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_28_399s61_CACT.fq.bz2 dm_77h_28_399s62_CACT.fq.bz2,fastq fastq,731276158.0,3620179.0,GSM1129619 r1,0:101 1:101,A:173989639;C:199270484;G:188281318;T:169725601;N:9116,101,101,,,173989639,199270484,188281318,169725601,9116,SRX271957,SRS416253,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",2,0.84121,0.79968,0.02287,0.02141,0.89658,0.89051,0.63318,0.63061,101,101,B,B,biological fallback assumption,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36719,SRR835160,SRX271956,SRS416252,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 28 436,GSM1129618,,tissue:melanocytes|hpf,dm 77h 28 436,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129618,GSM1129618: dm 77h 28 436; Danio rerio; RNA Seq,GSM1129618 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129618,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_28_436_CACT.fq.bz2,fastq,214752678.0,5113159.0,GSM1129618 r1,0:42,A:50210098;C:62230664;G:49216487;T:52926929;N:168500,42,,,,50210098,62230664,49216487,52926929,168500,SRX271956,SRS416252,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.80956,,0.02412,,0.87024,,0.62475,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36720,SRR835159,SRX271955,SRS416251,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 28 351,GSM1129617,,tissue:melanocytes|hpf,dm 77h 28 351,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129617,GSM1129617: dm 77h 28 351; Danio rerio; RNA Seq,GSM1129617 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129617,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_28_351_CACT.fq.bz2,fastq,84584556.0,2349571.0,GSM1129617 r1,0:36,A:19843286;C:24761303;G:18855233;T:21124051;N:683,36,,,,19843286,24761303,18855233,21124051,683,SRX271955,SRS416251,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.75246,,0.02663,,0.87054,,0.59024,,36,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36721,SRR835158,SRX271954,SRS416250,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 27 436,GSM1129616,,tissue:melanocytes|hpf,dm 77h 27 436,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129616,GSM1129616: dm 77h 27 436; Danio rerio; RNA Seq,GSM1129616 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129616,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_27_436_TTAT.fq.bz2,fastq,194114466.0,4621773.0,GSM1129616 r1,0:42,A:44579558;C:47759429;G:44539735;T:57080409;N:155335,42,,,,44579558,47759429,44539735,57080409,155335,SRX271954,SRS416250,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.80787,,0.04019,,0.86263,,0.57373,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36722,SRR835157,SRX271953,SRS416249,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 27 351,GSM1129615,,tissue:melanocytes|hpf,dm 77h 27 351,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129615,GSM1129615: dm 77h 27 351; Danio rerio; RNA Seq,GSM1129615 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129615,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_27_351_TTAT.fq.bz2,fastq,113087304.0,3141314.0,GSM1129615 r1,0:36,A:26171623;C:27323878;G:24871328;T:34719586;N:889,36,,,,26171623,27323878,24871328,34719586,889,SRX271953,SRS416249,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.75375,,0.04337,,0.85878,,0.5311,,36,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36723,SRR835156,SRX271952,SRS416248,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 26 433,GSM1129614,,tissue:melanocytes|hpf,dm 77h 26 433,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129614,GSM1129614: dm 77h 26 433; Danio rerio; RNA Seq,GSM1129614 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129614,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_26_433_AGAT.fq.bz2,fastq,34005804.0,809662.0,GSM1129614 r1,0:42,A:8766789;C:8066807;G:8338043;T:8833768;N:397,42,,,,8766789,8066807,8338043,8833768,397,SRX271952,SRS416248,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.81802,,0.05684,,0.88787,,0.56756,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36724,SRR835155,SRX271951,SRS416247,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 26 351,GSM1129613,,tissue:melanocytes|hpf,dm 77h 26 351,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129613,GSM1129613: dm 77h 26 351; Danio rerio; RNA Seq,GSM1129613 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129613,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_26_351_AGAT.fq.bz2,fastq,101340432.0,2815012.0,GSM1129613 r1,0:36,A:26592120;C:23518331;G:24503636;T:26725495;N:850,36,,,,26592120,23518331,24503636,26725495,850,SRX271951,SRS416247,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.75158,,0.05787,,0.88986,,0.57167,,36,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36725,SRR835154,SRX271950,SRS416245,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 25 399s62,GSM1129612,,tissue:melanocytes|hpf,dm 77h 25 399s62,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129612,GSM1129612: dm 77h 25 399s62; Danio rerio; RNA Seq,GSM1129612 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129612,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,,,1715809008.0,8494104.0,GSM1129612 r1,0:101 1:101,A:405310561;C:444474389;G:467897835;T:398105078;N:21145,101,101,,,405310561,444474389,467897835,398105078,21145,SRX271950,SRS416245,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",2,0.66327,0.58026,0.07911,0.07288,0.91721,0.9083,0.56907,0.5363,101,101,B,B,biological fallback assumption,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36726,SRR835153,SRX271949,SRS416246,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 25 399s61,GSM1129611,,tissue:melanocytes|hpf,dm 77h 25 399s61,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129611,GSM1129611: dm 77h 25 399s61; Danio rerio; RNA Seq,GSM1129611 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129611,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_25_399s61_TCTT.fq.bz2 dm_77h_25_399s62_TCTT.fq.bz2,fastq fastq,1715809008.0,8494104.0,GSM1129611 r1,0:101 1:101,A:405310561;C:444474389;G:467897835;T:398105078;N:21145,101,101,,,405310561,444474389,467897835,398105078,21145,SRX271949,SRS416246,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",2,0.66325,0.58027,0.0792,0.07327,0.91741,0.90847,0.56374,0.53632,101,101,B,B,biological fallback assumption,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36727,SRR835152,SRX271948,SRS416244,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 25 433,GSM1129610,,tissue:melanocytes|hpf,dm 77h 25 433,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129610,GSM1129610: dm 77h 25 433; Danio rerio; RNA Seq,GSM1129610 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129610,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_25_433_TCTT.fq.bz2,fastq,489386604.0,11652062.0,GSM1129610 r1,0:42,A:99597908;C:134563195;G:119005661;T:136214210;N:5630,42,,,,99597908,134563195,119005661,136214210,5630,SRX271948,SRS416244,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.79072,,0.08396,,0.87497,,0.51318,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36728,SRR835151,SRX271947,SRS416243,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 77h 25 351,GSM1129609,,tissue:melanocytes|hpf,dm 77h 25 351,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129609,GSM1129609: dm 77h 25 351; Danio rerio; RNA Seq,GSM1129609 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129609,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_77h_25_351_TCTT.fq.bz2,fastq,98765532.0,2743487.0,GSM1129609 r1,0:36,A:19387910;C:27338385;G:23220852;T:28817575;N:810,36,,,,19387910,27338385,23220852,28817575,810,SRX271947,SRS416243,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.75369,,0.08166,,0.87667,,0.51755,,36,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36729,SRR835150,SRX271946,SRS416242,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 58h 45 440,GSM1129608,,tissue:melanocytes|hpf,dm 58h 45 440,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129608,GSM1129608: dm 58h 45 440; Danio rerio; RNA Seq,GSM1129608 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129608,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_58h_45_440.fq.bz2,fastq,203660982.0,4849071.0,GSM1129608 r1,0:42,A:46576136;C:50204145;G:47522343;T:59345685;N:12673,42,,,,46576136,50204145,47522343,59345685,12673,SRX271946,SRS416242,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.86133,,0.0978,,0.84565,,0.53118,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36730,SRR835149,SRX271945,SRS416241,SRP021517,PRJNA198908,Gene Expression Analysis of Zebrafish Melanocytes  Iridophores  and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin,GSE46387,Transcriptome Analysis,In order to facilitate understanding of pigment cell biology  we developed a method to concomitantly purify melanocytes  iridophores  and retinal pigmented epithelium from zebrafish  and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co enrichment in melanocytes and retinal pigmented epithelium  as well as in melanocytes and iridophores.  We found 214 genes co enriched in melanocytes and retinal pigmented epithelium  indicating the shared functions of melanin producing cells.  We found 62 genes significantly co enriched in melanocytes and iridophores  illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate limiting substrate for purine synthesis  phosphoribosyl pyrophosphate  thus constituting a guanine cycle.  The purification procedure and expression analysis described here  along with the accompanying transcriptome wide expression data  provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types  and will be a useful resource for further studies of pigment cell biology. Overall design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing,,pubmed:23874447,,dm 58h 23 351,GSM1129607,,tissue:melanocytes|hpf,dm 58h 23 351,Align sequences using Novoalign to cDNA database Calculate RPKMs from Novoalign output using Perl Genome build: Non redundant database of 25 102 cDNAs generated by Higdon et al 2013  based on NCBI's RefSeq build from 8/29/2012  current RefSeq available at ftp://ftp.ncbi.nlm.nih.gov/refseq/D rerio/mRNA Prot/zebrafish.rna.fna.gz Supplementary files format and content: Excell file containing RPKMs of each library,melanocytes,,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,,hpf,GSM1129607,GSM1129607: dm 58h 23 351; Danio rerio; RNA Seq,GSM1129607 1,,1,This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.  Zebrafish were reared and bred according to standard protocols.  The fish used in this study were homozygous for a temperature sensitive allele of micropthalmia transcription factor  mitfavc7.  This mutant facilitated the collection of RPE  as mitfa is not required for RPE development in zebrafish.  Melanocytes  iridophores  and RPE develop normally at 25°C in mitfavc7.  When held at 32°C  the neural crest derived melanocytes do not develop  but RPE and iridophores develop normally.  All melanocyte and iridophore samples were incubated at 25°C prior to collection.  Fish were anesthetized with Tricaine  rinsed with Ca   Mg  DPBS Sigma  D8537  and immersed in 100mL TrypLE Express Invitrogen  12604039 per 1000 fish.  Fish were incubated at 37°C and shaken at 100rpm for 15 20 minutes  followed by trituration with a Pasteur pipette to remove eyes from larva.  post separation of eyes and larva  each group was placed in TrypLE Express and shaken at 100rpm at 37°C for 1 1.5 hr.  Dissociated cells were filtered through a 120uM screen into 50 mL tubes.  Remaining intact tissue was triturated 10 20 times  and again filtered through a 120uM screen into the dissociated cells.  Dissociated cells were pelleted in a swinging bucket rotor Eppendorf 5810 R at 500 relative centrifugal force rcf for 5 minutes at 4°C  then resuspended in 1mL cold isotonic Percoll Sigma  P1644 by gentle pipetting.  Isotonic Percoll was prepared by mixing 1 part 10X PBS with 9 parts Percoll.  Resuspended cells were transferred to 1.6mL Eppendorf tubes and spun at 2000rcf for 5 minutes at 4°C in a swinging bucket rotor for isopycnic separation.  Pigment cells in the pellet were then resuspended in 400µL of ice cold DPBS with 2% fetal calf serum FCS  and placed onto preformed Percoll density gradients.  Preformed gradients were prepared via centrifugation of 1mL aliquots of isotonic Percoll in 1.6mL tubes at 10 000rcf for 15 minutes at 4°C in a fixed angle micro centrifuge Eppendorf 5415 R.  Tubes containing preformed Percoll gradients with overlying cell suspensions were centrifuged in a swinging bucket rotor at 2000rcf for 10 minutes at 4°C.  Following centrifugation  overlying Percoll was aspirated  leaving the final 100µL containing the pigment cell pellet.  Cells were resuspended with 50µL of cold DPBS with 2% FCS and transferred to a clean 1.6mL tube containing 500µL of cold DPBS with 2% FCS  and kept on ice until mRNA extraction or FACS.      FACS   We used the inherent properties of the pigmented cells to perform Fluorescence Activated Cell Sorting FACS.  Following resuspension in 500µL cold DPBS with 2% FCS  the enriched cell populations were screened through a 30uM cell filter Partec  04 0042 2316.  Cells were analyzed and sorted with a Dako MoFlo cell sorter using a 120uM nozzle at a drop drive DD frequency of 22390Hz.  Cells were illuminated using a 488 nm laser.  Cells were gated on two attributes to separate cells from each other and from cellular debris.  Cellular debris was detected using forward and side scatter  selecting against the smallest particles 1µm or less.  Cells were sorted based on detection using 510 530nm and 575 595nm filters  corresponding to FL1 and FL2 in Figure 1D  respectively.  When excited by the 488 nm laser  the autofluorescence of iridophores is clearly detectable in these channels as a group of cells extending at a 45 degree line in the upper right quadrant.  Melanocytes and RPE do not autofluoresce with this intensity when excited by the 488nm laser  and cluster at the lower left of the FACS plot.  Cells were collected into ice cold DPBS with 2% FCS and kept on ice until mRNA extraction. Pigment cell cDNA library construction was as follows.  For mRNA extraction the Dynabeads® mRNA DIRECT Kit Invitrogen was used per manufacturer's instructions.  Following mRNA elution from the Dynabeads  first strand cDNA synthesis was performed using MMLV reverse transcriptase Clontech using an anchored polyT primer tailed with a universal primer sequence See Table S3 for primer sequences and Figure 2 for pigment cell cDNA library construction overview.  A universal primer sequence was also added to the three prime end of the first strand by template switching  allowing for PCR amplification of the resultant cDNA  [43 44].  Following PCR amplification using the high fidelity polymerase LA Taq TaKaRa   PCR cycle: 95C for 1 minute  followed by 20 cycles of 98C for 25 seconds  60C for 1 minute  68C for 20 minutes  cDNA was digested with AluI and RsaI restriction enzymes NEB.  Blunt end enzymatic fragmentation of cDNA was used instead of sonication and gel extraction to minimize loss of sample material and eliminate the end repair step of Illumina library preparation.  Since this reduced representation strategy might miss short cDNAs that lack both restriction sites  we sought to avoid this by including enzyme recognition sites within the cDNA amplification primers.  This allows for the inclusion of short cDNAs in our libraries.  Standard Illumina library preparations followed  performed by the Genome Technology Access Center GTAC at Washington University in St. Louis http://gtac.wustl.edu.  In brief  a single A was added to the 3’ end of each strand  Y adapters ligated  and library enrichment PCR performed  followed by gel extraction size selection for fragments ranging from 200 400 base pairs in length.  Illumina library construction of pooled 3dpf embryos was performed by GTAC from total RNA extracted with Trizol reagent as previously described [45].  No PCR amplification of whole embryo cDNA was performed prior to Illumina adapter ligation and library enrichment.  Sequencing was performed on the GAIIX Illumina platform.,GEO Accession:GSM1129607,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,dm_58h_23_351_GAAT.fq.bz2,fastq,106123464.0,2947874.0,GSM1129607 r1,0:36,A:26495114;C:26882652;G:25518440;T:27226454;N:804,36,,,,26495114,26882652,25518440,27226454,804,SRX271945,SRS416241,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.78561,,0.06331,,0.85107,,0.46073,,36,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Skin,Surface Structure
36741,SRR850608,SRX277695,SRS420758,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr18 danrer skin 42months rep5,GSM1141118,,source name:total RNA extracted from skin|strain:TuAB|age:42 month|tissue:skin,Nr18 danrer skin 42months rep5,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:42 month|tissue:skin,GSM1141118,GSM1141118: Nr18 danrer skin 42months rep5; Danio rerio; RNA Seq,GSM1141118 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141118,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr18_3.5y_l3_export.fq.gz,fastq,3269418540.0,43018665.0,GSM1141118 r1,0:76,A:892486836;C:744426899;G:749230539;T:883114979;N:159287,76,,,,892486836,744426899,749230539,883114979,159287,SRX277695,SRS420758,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91402,,0.1106,,0.73436,,0.4911,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36742,SRR850607,SRX277694,SRS420757,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr14 danrer skin 42months rep4,GSM1141117,,source name:total RNA extracted from skin|strain:TuAB|age:42 month|tissue:skin,Nr14 danrer skin 42months rep4,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:42 month|tissue:skin,GSM1141117,GSM1141117: Nr14 danrer skin 42months rep4; Danio rerio; RNA Seq,GSM1141117 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141117,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr14_3.5y_l2_export.fq.gz,fastq,2300890044.0,30274869.0,GSM1141117 r1,0:76,A:631736236;C:525065199;G:510761115;T:633208038;N:119456,76,,,,631736236,525065199,510761115,633208038,119456,SRX277694,SRS420757,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.90718,,0.11607,,0.71997,,0.49141,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36743,SRR850606,SRX277693,SRS420790,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr13 danrer skin 42months rep3,GSM1141116,,source name:total RNA extracted from skin|strain:TuAB|age:42 month|tissue:skin,Nr13 danrer skin 42months rep3,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:42 month|tissue:skin,GSM1141116,GSM1141116: Nr13 danrer skin 42months rep3; Danio rerio; RNA Seq,GSM1141116 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141116,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr13_3.5y_l1_export.fastq.gz,fastq,3508110372.0,46159347.0,GSM1141116 r1,0:76,A:959986155;C:782258637;G:803256847;T:962450623;N:158110,76,,,,959986155,782258637,803256847,962450623,158110,SRX277693,SRS420790,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.85747,,0.12068,,0.73819,,0.4917,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36744,SRR850605,SRX277692,SRS420789,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr12 danrer skin 42months rep2,GSM1141115,,source name:total RNA extracted from skin|strain:TuAB|age:42 month|tissue:skin,Nr12 danrer skin 42months rep2,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:42 month|tissue:skin,GSM1141115,GSM1141115: Nr12 danrer skin 42months rep2; Danio rerio; RNA Seq,GSM1141115 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141115,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr12_3.5y_l3_export.fq.gz,fastq,3961243804.0,52121629.0,GSM1141115 r1,0:76,A:1092811778;C:874440215;G:892629531;T:1099367104;N:1995176,76,,,,1092811778,874440215,892629531,1099367104,1995176,SRX277692,SRS420789,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.88854,,0.12633,,0.73746,,0.53849,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36745,SRR850604,SRX277691,SRS420788,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr11 danrer skin 42months rep1,GSM1141114,,source name:total RNA extracted from skin|strain:TuAB|age:42 month|tissue:skin,Nr11 danrer skin 42months rep1,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:42 month|tissue:skin,GSM1141114,GSM1141114: Nr11 danrer skin 42months rep1; Danio rerio; RNA Seq,GSM1141114 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141114,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr11_3.5y_l2_export.fq.gz,fastq,3327777040.0,43786540.0,GSM1141114 r1,0:76,A:920364748;C:745739060;G:758842746;T:901558784;N:1271702,76,,,,920364748,745739060,758842746,901558784,1271702,SRX277691,SRS420788,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.86781,,0.11618,,0.72888,,0.50217,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36746,SRR850603,SRX277690,SRS420756,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr76 danrer skin 24months rep6,GSM1141113,,source name:total RNA extracted from skin|strain:TuAB|age:24 month|tissue:skin,Nr76 danrer skin 24months rep6,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:24 month|tissue:skin,GSM1141113,GSM1141113: Nr76 danrer skin 24months rep6; Danio rerio; RNA Seq,GSM1141113 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141113,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr76_2y.fastq.gz,fastq,2234765940.0,29404815.0,GSM1141113 r1,0:76,A:582794172;C:524083367;G:547601836;T:579473821;N:812744,76,,,,582794172,524083367,547601836,579473821,812744,SRX277690,SRS420756,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.89808,,0.07989,,0.73511,,0.47456,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36747,SRR850602,SRX277689,SRS420755,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr75 danrer skin 24months rep5,GSM1141112,,source name:total RNA extracted from skin|strain:TuAB|age:24 month|tissue:skin,Nr75 danrer skin 24months rep5,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:24 month|tissue:skin,GSM1141112,GSM1141112: Nr75 danrer skin 24months rep5; Danio rerio; RNA Seq,GSM1141112 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141112,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr75_2y.fastq.gz,fastq,1000606196.0,13165871.0,GSM1141112 r1,0:76,A:270569436;C:233353660;G:238144829;T:258045669;N:492602,76,,,,270569436,233353660,238144829,258045669,492602,SRX277689,SRS420755,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.8949,,0.07966,,0.7293,,0.47729,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36748,SRR850601,SRX277688,SRS420754,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr73 danrer skin 24months rep4,GSM1141111,,source name:total RNA extracted from skin|strain:TuAB|age:24 month|tissue:skin,Nr73 danrer skin 24months rep4,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:24 month|tissue:skin,GSM1141111,GSM1141111: Nr73 danrer skin 24months rep4; Danio rerio; RNA Seq,GSM1141111 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141111,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr73_2y.fastq.gz,fastq,2283103688.0,30040838.0,GSM1141111 r1,0:76,A:601138588;C:527906584;G:549863593;T:603391353;N:803570,76,,,,601138588,527906584,549863593,603391353,803570,SRX277688,SRS420754,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.90138,,0.08154,,0.72598,,0.49455,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36749,SRR850600,SRX277687,SRS420787,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr72 danrer skin 24months rep3,GSM1141110,,source name:total RNA extracted from skin|strain:TuAB|age:24 month|tissue:skin,Nr72 danrer skin 24months rep3,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:24 month|tissue:skin,GSM1141110,GSM1141110: Nr72 danrer skin 24months rep3; Danio rerio; RNA Seq,GSM1141110 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141110,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr72_2y.fastq.gz,fastq,1692545916.0,22270341.0,GSM1141110 r1,0:76,A:447927627;C:393400289;G:405212681;T:445305247;N:700072,76,,,,447927627,393400289,405212681,445305247,700072,SRX277687,SRS420787,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.88704,,0.08604,,0.72833,,0.47215,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36750,SRR850599,SRX277686,SRS420786,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr71 danrer skin 24months rep2,GSM1141109,,source name:total RNA extracted from skin|strain:TuAB|age:24 month|tissue:skin,Nr71 danrer skin 24months rep2,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:24 month|tissue:skin,GSM1141109,GSM1141109: Nr71 danrer skin 24months rep2; Danio rerio; RNA Seq,GSM1141109 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141109,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr71_2y.fastq.gz,fastq,1897129404.0,24962229.0,GSM1141109 r1,0:76,A:500740954;C:442627409;G:457159802;T:495689680;N:911559,76,,,,500740954,442627409,457159802,495689680,911559,SRX277686,SRS420786,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.88731,,0.07537,,0.73628,,0.4846,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36751,SRR850598,SRX277685,SRS420785,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr70 danrer skin 24months rep1,GSM1141108,,source name:total RNA extracted from skin|strain:TuAB|age:24 month|tissue:skin,Nr70 danrer skin 24months rep1,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:24 month|tissue:skin,GSM1141108,GSM1141108: Nr70 danrer skin 24months rep1; Danio rerio; RNA Seq,GSM1141108 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141108,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr70_2y.fastq.gz,fastq,2239190508.0,29463033.0,GSM1141108 r1,0:76,A:599665392;C:514113743;G:531871299;T:592730477;N:809597,76,,,,599665392,514113743,531871299,592730477,809597,SRX277685,SRS420785,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.89167,,0.09031,,0.73515,,0.49129,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36752,SRR850597,SRX277684,SRS420784,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr30 danrer skin 5months rep9,GSM1141107,,source name:total RNA extracted from skin|strain:TuAB|age:5 month|tissue:skin,Nr30 danrer skin 5months rep9,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:5 month|tissue:skin,GSM1141107,GSM1141107: Nr30 danrer skin 5months rep9; Danio rerio; RNA Seq,GSM1141107 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141107,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr30_5m.fastq.gz,fastq,2502725752.0,32930602.0,GSM1141107 r1,0:76,A:684745428;C:570860380;G:565883575;T:679750149;N:1486220,76,,,,684745428,570860380,565883575,679750149,1486220,SRX277684,SRS420784,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91789,,0.10423,,0.7263,,0.4909,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36753,SRR850596,SRX277683,SRS420783,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr29 danrer skin 5months rep8,GSM1141106,,source name:total RNA extracted from skin|strain:TuAB|age:5 month|tissue:skin,Nr29 danrer skin 5months rep8,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:5 month|tissue:skin,GSM1141106,GSM1141106: Nr29 danrer skin 5months rep8; Danio rerio; RNA Seq,GSM1141106 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141106,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr29_5m.fastq.gz,fastq,2012479468.0,26479993.0,GSM1141106 r1,0:76,A:558800574;C:450898608;G:447907004;T:553773824;N:1099458,76,,,,558800574,450898608,447907004,553773824,1099458,SRX277683,SRS420783,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.9055,,0.1197,,0.7119,,0.48553,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36754,SRR850595,SRX277682,SRS420782,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr28 danrer skin 5months rep7,GSM1141105,,source name:total RNA extracted from skin|strain:TuAB|age:5 month|tissue:skin,Nr28 danrer skin 5months rep7,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:5 month|tissue:skin,GSM1141105,GSM1141105: Nr28 danrer skin 5months rep7; Danio rerio; RNA Seq,GSM1141105 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141105,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr28_5m.fastq.gz,fastq,2075098224.0,27303924.0,GSM1141105 r1,0:76,A:555062827;C:487427075;G:487360186;T:544381395;N:866741,76,,,,555062827,487427075,487360186,544381395,866741,SRX277682,SRS420782,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91784,,0.08866,,0.73208,,0.47793,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36755,SRR850594,SRX277681,SRS420781,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr27 danrer skin 5months rep6,GSM1141104,,source name:total RNA extracted from skin|strain:TuAB|age:5 month|tissue:skin,Nr27 danrer skin 5months rep6,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:5 month|tissue:skin,GSM1141104,GSM1141104: Nr27 danrer skin 5months rep6; Danio rerio; RNA Seq,GSM1141104 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141104,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr27_5m.fastq.gz,fastq,2415354480.0,31780980.0,GSM1141104 r1,0:76,A:661012615;C:552471296;G:546304105;T:654315033;N:1251431,76,,,,661012615,552471296,546304105,654315033,1251431,SRX277681,SRS420781,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.91282,,0.104,,0.7236,,0.48186,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36756,SRR850593,SRX277680,SRS420780,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr26 danrer skin 5months rep5,GSM1141103,,source name:total RNA extracted from skin|strain:TuAB|age:5 month|tissue:skin,Nr26 danrer skin 5months rep5,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:5 month|tissue:skin,GSM1141103,GSM1141103: Nr26 danrer skin 5months rep5; Danio rerio; RNA Seq,GSM1141103 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141103,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr26_5m_l8_export.fq.gz,fastq,1866135464.0,24554414.0,GSM1141103 r1,0:76,A:495417130;C:442746432;G:436942898;T:490685510;N:343494,76,,,,495417130,442746432,436942898,490685510,343494,SRX277680,SRS420780,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92443,,0.08023,,0.7288,,0.48902,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36757,SRR850592,SRX277679,SRS420778,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr25 danrer skin 5months rep4,GSM1141102,,source name:total RNA extracted from skin|strain:TuAB|age:5 month|tissue:skin,Nr25 danrer skin 5months rep4,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:5 month|tissue:skin,GSM1141102,GSM1141102: Nr25 danrer skin 5months rep4; Danio rerio; RNA Seq,GSM1141102 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141102,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr25_5m_l7_export.fq.gz,fastq,2146470508.0,28243033.0,GSM1141102 r1,0:76,A:583770580;C:491677255;G:491536604;T:579365742;N:120327,76,,,,583770580,491677255,491536604,579365742,120327,SRX277679,SRS420778,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.89568,,0.11303,,0.71778,,0.47882,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36758,SRR850591,SRX277678,SRS420779,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr23 danrer skin 5months rep3,GSM1141101,,source name:total RNA extracted from skin|strain:TuAB|age:5 month|tissue:skin,Nr23 danrer skin 5months rep3,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:5 month|tissue:skin,GSM1141101,GSM1141101: Nr23 danrer skin 5months rep3; Danio rerio; RNA Seq,GSM1141101 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141101,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr23_5m_l6_export.fq.gz,fastq,2628691268.0,34588043.0,GSM1141101 r1,0:76,A:693944705;C:617538873;G:611502862;T:705582669;N:122159,76,,,,693944705,617538873,611502862,705582669,122159,SRX277678,SRS420779,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92203,,0.09747,,0.71378,,0.47796,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36759,SRR850590,SRX277677,SRS420777,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr22 danrer skin 5months rep2,GSM1141100,,source name:total RNA extracted from skin|strain:TuAB|age:5 month|tissue:skin,Nr22 danrer skin 5months rep2,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:5 month|tissue:skin,GSM1141100,GSM1141100: Nr22 danrer skin 5months rep2; Danio rerio; RNA Seq,GSM1141100 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141100,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr22_5m.fastq.gz,fastq,3561382724.0,46860299.0,GSM1141100 r1,0:76,A:956105654;C:826498432;G:826128735;T:952426883;N:223020,76,,,,956105654,826498432,826128735,952426883,223020,SRX277677,SRS420777,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.93192,,0.09055,,0.72876,,0.52416,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
36760,SRR850589,SRX277676,SRS420776,SRP022612,PRJNA203029,RNA seq of Danio rerio and Mus musculus skin for three different age groups,GSE46916,Other,Background: Comparison of temporal gene expression profiles. The RNA seq data comprises 3 age groups: 2  15 month and 30 month for mouse skin; 5  24 month and 42 month for zebrafish skin. Illumina 50bp single stranded single read RNA sequencing Jena Centre for Systems Biology of Ageing   JenAge www.jenage.de Overall design: 15 samples for mouse: 5 biological replicates for 2 month  6 biological replicates for 15 month and 4 biological replicates for 30 month; 20 samples for zebrafish: 9 biological replicates for 5 month  6 biological replicates for 24 month and 5 biological replicates for 42 month,,pubmed:26620638,,Nr21 danrer skin 5months rep1,GSM1141099,,source name:total RNA extracted from skin|strain:TuAB|age:5 month|tissue:skin,Nr21 danrer skin 5months rep1,Illumina Casava software used for extraction of FASTQ files FASTQ files were mapped using Bowtie vs. the respective genomic sequence and a splice site data set created using UCSC's RefSeq annotation for each species Counting of uniquely mapped reads and assignment to RefSeq transcripts/genes using R Statistical Language and Bioconductor Calculation of RPKM values for each transcript and gene from the corresponding RefSeq annotation Genome build: D. rerio: danRer7; M. musculus: mm9 Supplementary files format and content: Excel file include raw counts and RPKM values for each sample,total RNA extracted from skin,,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,,strain:TuAB|age:5 month|tissue:skin,GSM1141099,GSM1141099: Nr21 danrer skin 5months rep1; Danio rerio; RNA Seq,GSM1141099 1,,1,D. rerio: Skin tissue from male zebrafish was dissected and stored in RNAlater Qiagen  Hilden  Germany at 80 °C. Total RNA was isolated with TRIzol Life Technologies  Darmstadt  Germany according to the instructions of the manufacturer. M. musculus: The isolated skin was homogenized in 500 μl QIAzol Qiagen by subsequently adding 100 μl chloroform. Following phase separation  the aqueous phase was transferred into a fresh tube  then 0.16 volume NaAc 2 M  pH 4.0 and 1.1 volume isopropanol were added. The RNA was precipitated by centrifugation and the pellet was washed with 75% ethanol. Total RNA was resuspended in 20 μl water GIBCO and stored at –80°C until use. D. rerio: 5 µg of total RNA was used for preparation of libraries using Illumina’s mRNA Seq sample prep kit Illumina  San Diego  CA  USA following the manufacturer’s instruction. M. musculus: 2.5 µg of total RNA was used with Illumina’s TruSeq RNA sample prep kit.,GEO Accession:GSM1141099,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina Genome Analyzer IIx,,SRP022612,,,Nr21_5m_l4_export.fq.gz,fastq,2709932304.0,35657004.0,GSM1141099 r1,0:76,A:730073083;C:631072920;G:619401848;T:729203191;N:181262,76,,,,730073083,631072920,619401848,729203191,181262,SRX277676,SRS420776,SRA076297,GEO,Leibniz Institute for Age Research - Fritz Lipmann Institute,1,0.92839,,0.10379,,0.71392,,0.4865,,76,,B,,usable mapping rate,illumina,early_illumina,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,Germany,2013-05-14,Adult,Adult,Skin,Surface Structure
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