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
10155,ERR5236196,ERX5039563,ERS5672247,ERP126773,PRJEB42852,RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,E-MTAB-10068,Transcriptome Analysis,Currently there is little knowledge on the functions and signaling pathways of MAB21L1. Therefore  we developed a zebrafish genetic model of mab21l1 deficiency to use in order to gain insight into possibly affected pathways and transcripts. We conducted RNA seq on zebrafish whole eyes which were dissected out at 5 dpf Three biological replicates per genotype wild type or homozygous mab21l1 c.107delA were collected  with 50 eyes per replicate. RNA was extracted and purified and submitted to Macrogen to conduct RNA Sequencing procedure.,ENA FIRST PUBLIC:2022 01 26|ENA LAST UPDATE:2022 01 26,,Protocols: Whole eyes were dissected from 5 dpf wild type or homozygous mab21l1 c.107delA embryos into 1X Ringer's solution. Three biological samples were collected per genotype  and each sample contained 50 eyes. To isolate RNA  whole eye samples were lysed and homogenized in TRI Reagent. Then  the Direct zol RNA MiniPrep Kit from ZymoResearch was followed to extract and purify RNA. RNA Samples were submitted to Macrogen for RNA Seq performance. Macrogen conducted library prep using a TruSeq Stranded mRNA LT Sample Prep Kit following the TruSeq Stranded mRNA Sample Preparation Guide  Part # 15031047 Rev. E.,Zebrafish WT 3,SAMEA7984965,"Department of Pediatrics and Cell Biology, Neurobiology and Anatomy, The Medical College of Wisconsin, Milwaukee, WI USA",ENA first public:2022 01 26|ENA last update:2022 01 26|External Id:SAMEA7984965|INSDC center alias:Department of Pediatrics and Cell Biology  Neurobiology and Anatomy  The Medical College of Wisconsin  Milwaukee  WI USA|INSDC center name:Department of Pediatrics and Cell Biology  Neurobiology and Anatomy  The Medical College of Wisconsin  Milwaukee  WI USA|INSDC first public:2022 01 26T00:16:15Z|INSDC last update:2022 01 26T00:16:15Z|INSDC status:public|Submitter Id:E MTAB 10068:Zebrafish WT 3|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:late embryonic stage|genotype:wild type genotype|organism part:eye|sample name:E MTAB 10068:Zebrafish WT 3,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing; RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,E MTAB 10068:Zebrafish WT 3 p,Zebrafish WT 3 p,RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,Whole eyes were dissected from 5 dpf wild type or homozygous mab21l1 c.107delA embryos into 1X Ringer's solution. Three biological samples were collected per genotype  and each sample contained 50 eyes. To isolate RNA  whole eye samples were  lysed and homogenized in TRI Reagent. Then  the Direct zol RNA MiniPrep Kit from ZymoResearch was followed to extract and purify RNA.  RNA Samples were submitted to Macrogen for RNA Seq performance. Macrogen conducted library prep using a TruSeq Stranded mRNA LT Sample Prep Kit following the TruSeq Stranded mRNA Sample Preparation Guide  Part # 15031047 Rev. E.,Experimental Factor: genotype:wild type genotype,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP126773,Illumina NovaSeq 6000 paired end sequencing; RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,ENA FIRST PUBLIC:2022 01 26|ENA LAST UPDATE:2022 01 26,Zebrafish_WT_3_1.fastq.gz Zebrafish_WT_3_2.fastq.gz,fastq fastq,7150981594.0,23678747.0,E MTAB 10068:Zebrafish WT 3 ,0:151 1:151,A:1885053833;C:1690814929;G:1772259046;T:1802798453;N:55333,151,151,,,1885053833,1690814929,1772259046,1802798453,55333,ERX5039563,ERS5672247,ERA3400155,"Department of Pediatrics and Cell Biology, Neurobiology and Anatomy, The Medical College of Wisconsin, Milwaukee, WI USA|European Nucleotide Archive","Department of Pediatrics and Cell Biology, Neurobiology and Anatomy, The Medical College of Wisconsin, Milwaukee, WI USA|European Nucleotide Archive",2,0.95124,0.95221,0.08338,0.08285,0.69181,0.69256,0.46157,0.46139,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2022-01-26,Larval,Larval,Eye,Sensory System
10156,ERR5236195,ERX5039562,ERS5672246,ERP126773,PRJEB42852,RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,E-MTAB-10068,Transcriptome Analysis,Currently there is little knowledge on the functions and signaling pathways of MAB21L1. Therefore  we developed a zebrafish genetic model of mab21l1 deficiency to use in order to gain insight into possibly affected pathways and transcripts. We conducted RNA seq on zebrafish whole eyes which were dissected out at 5 dpf Three biological replicates per genotype wild type or homozygous mab21l1 c.107delA were collected  with 50 eyes per replicate. RNA was extracted and purified and submitted to Macrogen to conduct RNA Sequencing procedure.,ENA FIRST PUBLIC:2022 01 26|ENA LAST UPDATE:2022 01 26,,Protocols: Whole eyes were dissected from 5 dpf wild type or homozygous mab21l1 c.107delA embryos into 1X Ringer's solution. Three biological samples were collected per genotype  and each sample contained 50 eyes. To isolate RNA  whole eye samples were lysed and homogenized in TRI Reagent. Then  the Direct zol RNA MiniPrep Kit from ZymoResearch was followed to extract and purify RNA. RNA Samples were submitted to Macrogen for RNA Seq performance. Macrogen conducted library prep using a TruSeq Stranded mRNA LT Sample Prep Kit following the TruSeq Stranded mRNA Sample Preparation Guide  Part # 15031047 Rev. E.,Zebrafish WT 2,SAMEA7984964,"Department of Pediatrics and Cell Biology, Neurobiology and Anatomy, The Medical College of Wisconsin, Milwaukee, WI USA",ENA first public:2022 01 26|ENA last update:2022 01 26|External Id:SAMEA7984964|INSDC center alias:Department of Pediatrics and Cell Biology  Neurobiology and Anatomy  The Medical College of Wisconsin  Milwaukee  WI USA|INSDC center name:Department of Pediatrics and Cell Biology  Neurobiology and Anatomy  The Medical College of Wisconsin  Milwaukee  WI USA|INSDC first public:2022 01 26T00:16:15Z|INSDC last update:2022 01 26T00:16:15Z|INSDC status:public|Submitter Id:E MTAB 10068:Zebrafish WT 2|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:late embryonic stage|genotype:wild type genotype|organism part:eye|sample name:E MTAB 10068:Zebrafish WT 2,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing; RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,E MTAB 10068:Zebrafish WT 2 p,Zebrafish WT 2 p,RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,Whole eyes were dissected from 5 dpf wild type or homozygous mab21l1 c.107delA embryos into 1X Ringer's solution. Three biological samples were collected per genotype  and each sample contained 50 eyes. To isolate RNA  whole eye samples were  lysed and homogenized in TRI Reagent. Then  the Direct zol RNA MiniPrep Kit from ZymoResearch was followed to extract and purify RNA.  RNA Samples were submitted to Macrogen for RNA Seq performance. Macrogen conducted library prep using a TruSeq Stranded mRNA LT Sample Prep Kit following the TruSeq Stranded mRNA Sample Preparation Guide  Part # 15031047 Rev. E.,Experimental Factor: genotype:wild type genotype,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP126773,Illumina NovaSeq 6000 paired end sequencing; RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,ENA FIRST PUBLIC:2022 01 26|ENA LAST UPDATE:2022 01 26,Zebrafish_WT_2_1.fastq.gz Zebrafish_WT_2_2.fastq.gz,fastq fastq,7214044026.0,23887563.0,E MTAB 10068:Zebrafish WT 2 ,0:151 1:151,A:1902219751;C:1705162605;G:1795933407;T:1810669383;N:58880,151,151,,,1902219751,1705162605,1795933407,1810669383,58880,ERX5039562,ERS5672246,ERA3400155,"Department of Pediatrics and Cell Biology, Neurobiology and Anatomy, The Medical College of Wisconsin, Milwaukee, WI USA|European Nucleotide Archive","Department of Pediatrics and Cell Biology, Neurobiology and Anatomy, The Medical College of Wisconsin, Milwaukee, WI USA|European Nucleotide Archive",2,0.95311,0.95386,0.07898,0.07845,0.69045,0.69061,0.46066,0.4628,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2022-01-26,Larval,Larval,Eye,Sensory System
10157,ERR5236194,ERX5039561,ERS5672245,ERP126773,PRJEB42852,RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,E-MTAB-10068,Transcriptome Analysis,Currently there is little knowledge on the functions and signaling pathways of MAB21L1. Therefore  we developed a zebrafish genetic model of mab21l1 deficiency to use in order to gain insight into possibly affected pathways and transcripts. We conducted RNA seq on zebrafish whole eyes which were dissected out at 5 dpf Three biological replicates per genotype wild type or homozygous mab21l1 c.107delA were collected  with 50 eyes per replicate. RNA was extracted and purified and submitted to Macrogen to conduct RNA Sequencing procedure.,ENA FIRST PUBLIC:2022 01 26|ENA LAST UPDATE:2022 01 26,,Protocols: Whole eyes were dissected from 5 dpf wild type or homozygous mab21l1 c.107delA embryos into 1X Ringer's solution. Three biological samples were collected per genotype  and each sample contained 50 eyes. To isolate RNA  whole eye samples were lysed and homogenized in TRI Reagent. Then  the Direct zol RNA MiniPrep Kit from ZymoResearch was followed to extract and purify RNA. RNA Samples were submitted to Macrogen for RNA Seq performance. Macrogen conducted library prep using a TruSeq Stranded mRNA LT Sample Prep Kit following the TruSeq Stranded mRNA Sample Preparation Guide  Part # 15031047 Rev. E.,Zebrafish WT 1,SAMEA7984963,"Department of Pediatrics and Cell Biology, Neurobiology and Anatomy, The Medical College of Wisconsin, Milwaukee, WI USA",ENA first public:2022 01 26|ENA last update:2022 01 26|External Id:SAMEA7984963|INSDC center alias:Department of Pediatrics and Cell Biology  Neurobiology and Anatomy  The Medical College of Wisconsin  Milwaukee  WI USA|INSDC center name:Department of Pediatrics and Cell Biology  Neurobiology and Anatomy  The Medical College of Wisconsin  Milwaukee  WI USA|INSDC first public:2022 01 26T00:16:15Z|INSDC last update:2022 01 26T00:16:15Z|INSDC status:public|Submitter Id:E MTAB 10068:Zebrafish WT 1|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:late embryonic stage|genotype:wild type genotype|organism part:eye|sample name:E MTAB 10068:Zebrafish WT 1,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing; RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,E MTAB 10068:Zebrafish WT 1 p,Zebrafish WT 1 p,RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,Whole eyes were dissected from 5 dpf wild type or homozygous mab21l1 c.107delA embryos into 1X Ringer's solution. Three biological samples were collected per genotype  and each sample contained 50 eyes. To isolate RNA  whole eye samples were  lysed and homogenized in TRI Reagent. Then  the Direct zol RNA MiniPrep Kit from ZymoResearch was followed to extract and purify RNA.  RNA Samples were submitted to Macrogen for RNA Seq performance. Macrogen conducted library prep using a TruSeq Stranded mRNA LT Sample Prep Kit following the TruSeq Stranded mRNA Sample Preparation Guide  Part # 15031047 Rev. E.,Experimental Factor: genotype:wild type genotype,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP126773,Illumina NovaSeq 6000 paired end sequencing; RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,ENA FIRST PUBLIC:2022 01 26|ENA LAST UPDATE:2022 01 26,Zebrafish_WT_1_1.fastq.gz Zebrafish_WT_1_2.fastq.gz,fastq fastq,6632043216.0,21960408.0,E MTAB 10068:Zebrafish WT 1 ,0:151 1:151,A:1763732625;C:1547925091;G:1636588945;T:1683739270;N:57285,151,151,,,1763732625,1547925091,1636588945,1683739270,57285,ERX5039561,ERS5672245,ERA3400155,"Department of Pediatrics and Cell Biology, Neurobiology and Anatomy, The Medical College of Wisconsin, Milwaukee, WI USA|European Nucleotide Archive","Department of Pediatrics and Cell Biology, Neurobiology and Anatomy, The Medical College of Wisconsin, Milwaukee, WI USA|European Nucleotide Archive",2,0.9495,0.95025,0.08958,0.08856,0.68661,0.68598,0.46029,0.46171,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2022-01-26,Larval,Larval,Eye,Sensory System
10158,ERR5236193,ERX5039560,ERS5672244,ERP126773,PRJEB42852,RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,E-MTAB-10068,Transcriptome Analysis,Currently there is little knowledge on the functions and signaling pathways of MAB21L1. Therefore  we developed a zebrafish genetic model of mab21l1 deficiency to use in order to gain insight into possibly affected pathways and transcripts. We conducted RNA seq on zebrafish whole eyes which were dissected out at 5 dpf Three biological replicates per genotype wild type or homozygous mab21l1 c.107delA were collected  with 50 eyes per replicate. RNA was extracted and purified and submitted to Macrogen to conduct RNA Sequencing procedure.,ENA FIRST PUBLIC:2022 01 26|ENA LAST UPDATE:2022 01 26,,Protocols: Whole eyes were dissected from 5 dpf wild type or homozygous mab21l1 c.107delA embryos into 1X Ringer's solution. Three biological samples were collected per genotype  and each sample contained 50 eyes. To isolate RNA  whole eye samples were lysed and homogenized in TRI Reagent. Then  the Direct zol RNA MiniPrep Kit from ZymoResearch was followed to extract and purify RNA. RNA Samples were submitted to Macrogen for RNA Seq performance. Macrogen conducted library prep using a TruSeq Stranded mRNA LT Sample Prep Kit following the TruSeq Stranded mRNA Sample Preparation Guide  Part # 15031047 Rev. E.,Zebrafish mab21l1 3,SAMEA7984962,"Department of Pediatrics and Cell Biology, Neurobiology and Anatomy, The Medical College of Wisconsin, Milwaukee, WI USA",ENA first public:2022 01 26|ENA last update:2022 01 26|External Id:SAMEA7984962|INSDC center alias:Department of Pediatrics and Cell Biology  Neurobiology and Anatomy  The Medical College of Wisconsin  Milwaukee  WI USA|INSDC center name:Department of Pediatrics and Cell Biology  Neurobiology and Anatomy  The Medical College of Wisconsin  Milwaukee  WI USA|INSDC first public:2022 01 26T00:16:15Z|INSDC last update:2022 01 26T00:16:15Z|INSDC status:public|Submitter Id:E MTAB 10068:Zebrafish mab21l1 3|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:late embryonic stage|genotype:homozygous mab21l1 c.107delA|organism part:eye|sample name:E MTAB 10068:Zebrafish mab21l1 3,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing; RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,E MTAB 10068:Zebrafish mab21l1 3 p,Zebrafish mab21l1 3 p,RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,Whole eyes were dissected from 5 dpf wild type or homozygous mab21l1 c.107delA embryos into 1X Ringer's solution. Three biological samples were collected per genotype  and each sample contained 50 eyes. To isolate RNA  whole eye samples were  lysed and homogenized in TRI Reagent. Then  the Direct zol RNA MiniPrep Kit from ZymoResearch was followed to extract and purify RNA.  RNA Samples were submitted to Macrogen for RNA Seq performance. Macrogen conducted library prep using a TruSeq Stranded mRNA LT Sample Prep Kit following the TruSeq Stranded mRNA Sample Preparation Guide  Part # 15031047 Rev. E.,Experimental Factor: genotype:homozygous mab21l1 c.107delA,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP126773,Illumina NovaSeq 6000 paired end sequencing; RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,ENA FIRST PUBLIC:2022 01 26|ENA LAST UPDATE:2022 01 26,Zebrafish_mab21l1_3_1.fastq.gz Zebrafish_mab21l1_3_2.fastq.gz,fastq fastq,6182833618.0,20472959.0,E MTAB 10068:Zebrafish mab21l1 3 ,0:151 1:151,A:1613191835;C:1471874932;G:1547131522;T:1550575662;N:59667,151,151,,,1613191835,1471874932,1547131522,1550575662,59667,ERX5039560,ERS5672244,ERA3400155,"Department of Pediatrics and Cell Biology, Neurobiology and Anatomy, The Medical College of Wisconsin, Milwaukee, WI USA|European Nucleotide Archive","Department of Pediatrics and Cell Biology, Neurobiology and Anatomy, The Medical College of Wisconsin, Milwaukee, WI USA|European Nucleotide Archive",2,0.95645,0.95788,0.0697,0.06969,0.70096,0.70033,0.43342,0.43242,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2022-01-26,Larval,Larval,Eye,Sensory System
10159,ERR5236192,ERX5039559,ERS5672243,ERP126773,PRJEB42852,RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,E-MTAB-10068,Transcriptome Analysis,Currently there is little knowledge on the functions and signaling pathways of MAB21L1. Therefore  we developed a zebrafish genetic model of mab21l1 deficiency to use in order to gain insight into possibly affected pathways and transcripts. We conducted RNA seq on zebrafish whole eyes which were dissected out at 5 dpf Three biological replicates per genotype wild type or homozygous mab21l1 c.107delA were collected  with 50 eyes per replicate. RNA was extracted and purified and submitted to Macrogen to conduct RNA Sequencing procedure.,ENA FIRST PUBLIC:2022 01 26|ENA LAST UPDATE:2022 01 26,,Protocols: Whole eyes were dissected from 5 dpf wild type or homozygous mab21l1 c.107delA embryos into 1X Ringer's solution. Three biological samples were collected per genotype  and each sample contained 50 eyes. To isolate RNA  whole eye samples were lysed and homogenized in TRI Reagent. Then  the Direct zol RNA MiniPrep Kit from ZymoResearch was followed to extract and purify RNA. RNA Samples were submitted to Macrogen for RNA Seq performance. Macrogen conducted library prep using a TruSeq Stranded mRNA LT Sample Prep Kit following the TruSeq Stranded mRNA Sample Preparation Guide  Part # 15031047 Rev. E.,Zebrafish mab21l1 2,SAMEA7984961,"Department of Pediatrics and Cell Biology, Neurobiology and Anatomy, The Medical College of Wisconsin, Milwaukee, WI USA",ENA first public:2022 01 26|ENA last update:2022 01 26|External Id:SAMEA7984961|INSDC center alias:Department of Pediatrics and Cell Biology  Neurobiology and Anatomy  The Medical College of Wisconsin  Milwaukee  WI USA|INSDC center name:Department of Pediatrics and Cell Biology  Neurobiology and Anatomy  The Medical College of Wisconsin  Milwaukee  WI USA|INSDC first public:2022 01 26T00:16:15Z|INSDC last update:2022 01 26T00:16:15Z|INSDC status:public|Submitter Id:E MTAB 10068:Zebrafish mab21l1 2|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:late embryonic stage|genotype:homozygous mab21l1 c.107delA|organism part:eye|sample name:E MTAB 10068:Zebrafish mab21l1 2,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing; RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,E MTAB 10068:Zebrafish mab21l1 2 p,Zebrafish mab21l1 2 p,RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,Whole eyes were dissected from 5 dpf wild type or homozygous mab21l1 c.107delA embryos into 1X Ringer's solution. Three biological samples were collected per genotype  and each sample contained 50 eyes. To isolate RNA  whole eye samples were  lysed and homogenized in TRI Reagent. Then  the Direct zol RNA MiniPrep Kit from ZymoResearch was followed to extract and purify RNA.  RNA Samples were submitted to Macrogen for RNA Seq performance. Macrogen conducted library prep using a TruSeq Stranded mRNA LT Sample Prep Kit following the TruSeq Stranded mRNA Sample Preparation Guide  Part # 15031047 Rev. E.,Experimental Factor: genotype:homozygous mab21l1 c.107delA,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP126773,Illumina NovaSeq 6000 paired end sequencing; RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,ENA FIRST PUBLIC:2022 01 26|ENA LAST UPDATE:2022 01 26,Zebrafish_mab21l1_2_1.fastq.gz Zebrafish_mab21l1_2_2.fastq.gz,fastq fastq,6989382602.0,23143651.0,E MTAB 10068:Zebrafish mab21l1 2 ,0:151 1:151,A:1841093814;C:1660652130;G:1754262273;T:1733319973;N:54412,151,151,,,1841093814,1660652130,1754262273,1733319973,54412,ERX5039559,ERS5672243,ERA3400155,"Department of Pediatrics and Cell Biology, Neurobiology and Anatomy, The Medical College of Wisconsin, Milwaukee, WI USA|European Nucleotide Archive","Department of Pediatrics and Cell Biology, Neurobiology and Anatomy, The Medical College of Wisconsin, Milwaukee, WI USA|European Nucleotide Archive",2,0.95595,0.95712,0.07199,0.07107,0.70485,0.70431,0.42033,0.42681,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2022-01-26,Larval,Larval,Eye,Sensory System
10160,ERR5236191,ERX5039558,ERS5672242,ERP126773,PRJEB42852,RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,E-MTAB-10068,Transcriptome Analysis,Currently there is little knowledge on the functions and signaling pathways of MAB21L1. Therefore  we developed a zebrafish genetic model of mab21l1 deficiency to use in order to gain insight into possibly affected pathways and transcripts. We conducted RNA seq on zebrafish whole eyes which were dissected out at 5 dpf Three biological replicates per genotype wild type or homozygous mab21l1 c.107delA were collected  with 50 eyes per replicate. RNA was extracted and purified and submitted to Macrogen to conduct RNA Sequencing procedure.,ENA FIRST PUBLIC:2022 01 26|ENA LAST UPDATE:2022 01 26,,Protocols: Whole eyes were dissected from 5 dpf wild type or homozygous mab21l1 c.107delA embryos into 1X Ringer's solution. Three biological samples were collected per genotype  and each sample contained 50 eyes. To isolate RNA  whole eye samples were lysed and homogenized in TRI Reagent. Then  the Direct zol RNA MiniPrep Kit from ZymoResearch was followed to extract and purify RNA. RNA Samples were submitted to Macrogen for RNA Seq performance. Macrogen conducted library prep using a TruSeq Stranded mRNA LT Sample Prep Kit following the TruSeq Stranded mRNA Sample Preparation Guide  Part # 15031047 Rev. E.,Zebrafish mab21l1 1,SAMEA7984960,"Department of Pediatrics and Cell Biology, Neurobiology and Anatomy, The Medical College of Wisconsin, Milwaukee, WI USA",ENA first public:2022 01 26|ENA last update:2022 01 26|External Id:SAMEA7984960|INSDC center alias:Department of Pediatrics and Cell Biology  Neurobiology and Anatomy  The Medical College of Wisconsin  Milwaukee  WI USA|INSDC center name:Department of Pediatrics and Cell Biology  Neurobiology and Anatomy  The Medical College of Wisconsin  Milwaukee  WI USA|INSDC first public:2022 01 26T00:16:15Z|INSDC last update:2022 01 26T00:16:15Z|INSDC status:public|Submitter Id:E MTAB 10068:Zebrafish mab21l1 1|age:5|broker name:ArrayExpress|common name:zebrafish|developmental stage:late embryonic stage|genotype:homozygous mab21l1 c.107delA|organism part:eye|sample name:E MTAB 10068:Zebrafish mab21l1 1,,,,,,,,,Illumina NovaSeq 6000 paired end sequencing; RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,E MTAB 10068:Zebrafish mab21l1 1 p,Zebrafish mab21l1 1 p,RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,Whole eyes were dissected from 5 dpf wild type or homozygous mab21l1 c.107delA embryos into 1X Ringer's solution. Three biological samples were collected per genotype  and each sample contained 50 eyes. To isolate RNA  whole eye samples were  lysed and homogenized in TRI Reagent. Then  the Direct zol RNA MiniPrep Kit from ZymoResearch was followed to extract and purify RNA.  RNA Samples were submitted to Macrogen for RNA Seq performance. Macrogen conducted library prep using a TruSeq Stranded mRNA LT Sample Prep Kit following the TruSeq Stranded mRNA Sample Preparation Guide  Part # 15031047 Rev. E.,Experimental Factor: genotype:homozygous mab21l1 c.107delA,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,ERP126773,Illumina NovaSeq 6000 paired end sequencing; RNA Seq of zebrafish whole eye comparing wild type and mab21l1 c.107delA p.Lys36Argfs*7 embryos at 5 dpf,ENA FIRST PUBLIC:2022 01 26|ENA LAST UPDATE:2022 01 26,Zebrafish_mab21l1_1_1.fastq.gz Zebrafish_mab21l1_1_2.fastq.gz,fastq fastq,6882371922.0,22789311.0,E MTAB 10068:Zebrafish mab21l1 1 ,0:151 1:151,A:1803380739;C:1630414039;G:1732263332;T:1716255495;N:58317,151,151,,,1803380739,1630414039,1732263332,1716255495,58317,ERX5039558,ERS5672242,ERA3400155,"Department of Pediatrics and Cell Biology, Neurobiology and Anatomy, The Medical College of Wisconsin, Milwaukee, WI USA|European Nucleotide Archive","Department of Pediatrics and Cell Biology, Neurobiology and Anatomy, The Medical College of Wisconsin, Milwaukee, WI USA|European Nucleotide Archive",2,0.95518,0.95612,0.07088,0.07032,0.70526,0.70457,0.42942,0.4302,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,unknown,unknown,,United States,2022-01-26,Larval,Larval,Eye,Sensory System
29107,SRR27151543,SRX22833143,SRS19813583,SRP476691,PRJNA1050282,Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf whole eye],GSE249751,Transcriptome Analysis,The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here  we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 /  retina  red cones are transformed to hybrid red/UV sensitive cones  green cones are transfated to blue cones  and the number of rods is greatly reduced. In the mouse Samd7 /  retina  dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether  Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones  thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf larvae  samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine  anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C  and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed  eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen  then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total  with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces.,parent bioproject:PRJNA1050288,pubmed:39531499,,5 dpf larval eyes  samd7 /   rep3,GSM7963641,,source name:eye|tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / |geo loc name:missing|collection date:missing,5 dpf larval eyes  samd7 /   rep3,Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next  Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression  DESeq2 1.34.0 was used in R 4.1.3  using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific  manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 /  adult retina Table S3  we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 /  and WT replicates  derived from 5dpf larval eyes. Genes are ranked by p adj.,eye,,To perform RNA seq on 5 dpf larvae  samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine  anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C  and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed  eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen  then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total  with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / ,GSM7963641,GSM7963641: 5 dpf larval eyes  samd7 /   rep3; Danio rerio; RNA Seq,GSM7963641 r1,GSM7963641,1,To perform RNA seq on 5 dpf larvae  samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine  anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C  and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed  eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen  then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total  with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP476691,,loader:fastq load.py,k7.AGGTATTCGG-GAGTCGCTTC.H37F5DSX2_AGGTATTCGG-GAGTCGCTTC_L003_R1.fastq.gz k7.AGGTATTCGG-GAGTCGCTTC.H37F5DSX2_AGGTATTCGG-GAGTCGCTTC_L003_R2.fastq.gz,fastq fastq,10747100652.0,35586426.0,GSM7963641 r1,0:151 1:151,A:2879574125;C:2414500329;G:2693618198;T:2759237631;N:170369,151,151,,,2879574125,2414500329,2693618198,2759237631,170369,SRX22833143,SRS19813583,SRA1765718,"Pathology and Immunology, Washington University School of Medicine","Pathology and Immunology, Washington University School of Medicine",2,0.95486,0.95989,0.05038,0.05167,0.73018,0.73438,0.41476,0.42743,151,151,B,B,biological fallback assumption,illumina,novaseq_era,3prime,random_priming,smarter,bulk,unknown,unknown,,United States,2023-12-08,Larval,Larval,Eye,Sensory System
29108,SRR27151544,SRX22833142,SRS19813582,SRP476691,PRJNA1050282,Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf whole eye],GSE249751,Transcriptome Analysis,The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here  we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 /  retina  red cones are transformed to hybrid red/UV sensitive cones  green cones are transfated to blue cones  and the number of rods is greatly reduced. In the mouse Samd7 /  retina  dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether  Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones  thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf larvae  samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine  anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C  and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed  eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen  then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total  with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces.,parent bioproject:PRJNA1050288,pubmed:39531499,,5 dpf larval eyes  samd7 /   rep2,GSM7963640,,source name:eye|tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / |geo loc name:missing|collection date:missing,5 dpf larval eyes  samd7 /   rep2,Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next  Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression  DESeq2 1.34.0 was used in R 4.1.3  using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific  manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 /  adult retina Table S3  we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 /  and WT replicates  derived from 5dpf larval eyes. Genes are ranked by p adj.,eye,,To perform RNA seq on 5 dpf larvae  samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine  anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C  and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed  eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen  then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total  with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / ,GSM7963640,GSM7963640: 5 dpf larval eyes  samd7 /   rep2; Danio rerio; RNA Seq,GSM7963640 r1,GSM7963640,1,To perform RNA seq on 5 dpf larvae  samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine  anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C  and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed  eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen  then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total  with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP476691,,loader:fastq load.py,k10.AATCGAACTC-TTAGACCTTC.H37F5DSX2_AATCGAACTC-TTAGACCTTC_L003_R1.fastq.gz k10.AATCGAACTC-TTAGACCTTC.H37F5DSX2_AATCGAACTC-TTAGACCTTC_L003_R2.fastq.gz,fastq fastq,10739126946.0,35560023.0,GSM7963640 r1,0:151 1:151,A:2888527520;C:2418475992;G:2670968816;T:2760983764;N:170854,151,151,,,2888527520,2418475992,2670968816,2760983764,170854,SRX22833142,SRS19813582,SRA1765718,"Pathology and Immunology, Washington University School of Medicine","Pathology and Immunology, Washington University School of Medicine",2,0.95306,0.95846,0.05658,0.05746,0.73269,0.73714,0.42191,0.43088,151,151,B,B,biological fallback assumption,illumina,novaseq_era,3prime,random_priming,smarter,bulk,unknown,unknown,,United States,2023-12-08,Larval,Larval,Eye,Sensory System
29109,SRR27151545,SRX22833141,SRS19813581,SRP476691,PRJNA1050282,Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf whole eye],GSE249751,Transcriptome Analysis,The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here  we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 /  retina  red cones are transformed to hybrid red/UV sensitive cones  green cones are transfated to blue cones  and the number of rods is greatly reduced. In the mouse Samd7 /  retina  dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether  Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones  thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf larvae  samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine  anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C  and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed  eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen  then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total  with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces.,parent bioproject:PRJNA1050288,pubmed:39531499,,5 dpf larval eyes  samd7 /   rep1,GSM7963639,,source name:eye|tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / |geo loc name:missing|collection date:missing,5 dpf larval eyes  samd7 /   rep1,Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next  Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression  DESeq2 1.34.0 was used in R 4.1.3  using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific  manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 /  adult retina Table S3  we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 /  and WT replicates  derived from 5dpf larval eyes. Genes are ranked by p adj.,eye,,To perform RNA seq on 5 dpf larvae  samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine  anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C  and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed  eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen  then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total  with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / ,GSM7963639,GSM7963639: 5 dpf larval eyes  samd7 /   rep1; Danio rerio; RNA Seq,GSM7963639 r1,GSM7963639,1,To perform RNA seq on 5 dpf larvae  samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine  anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C  and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed  eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen  then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total  with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP476691,,loader:fastq load.py,k11.CCGAAGATAA-TGGATTAACG.H37F5DSX2_CCGAAGATAA-TGGATTAACG_L003_R1.fastq.gz k11.CCGAAGATAA-TGGATTAACG.H37F5DSX2_CCGAAGATAA-TGGATTAACG_L003_R2.fastq.gz,fastq fastq,10514289154.0,34815527.0,GSM7963639 r1,0:151 1:151,A:2862247233;C:2377266250;G:2528917605;T:2745692826;N:165240,151,151,,,2862247233,2377266250,2528917605,2745692826,165240,SRX22833141,SRS19813581,SRA1765718,"Pathology and Immunology, Washington University School of Medicine","Pathology and Immunology, Washington University School of Medicine",2,0.95376,0.95966,0.05526,0.05681,0.72965,0.73273,0.42276,0.43148,151,151,B,B,biological fallback assumption,illumina,novaseq_era,3prime,random_priming,smarter,bulk,unknown,unknown,,United States,2023-12-08,Larval,Larval,Eye,Sensory System
29110,SRR27151546,SRX22833140,SRS19813578,SRP476691,PRJNA1050282,Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf whole eye],GSE249751,Transcriptome Analysis,The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here  we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 /  retina  red cones are transformed to hybrid red/UV sensitive cones  green cones are transfated to blue cones  and the number of rods is greatly reduced. In the mouse Samd7 /  retina  dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether  Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones  thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf larvae  samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine  anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C  and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed  eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen  then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total  with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces.,parent bioproject:PRJNA1050288,pubmed:39531499,,5 dpf larval eyes  WT  rep3,GSM7963638,,source name:eye|tissue:eye|developmental stage:5 dpf larvae|genotype:WT|geo loc name:missing|collection date:missing,5 dpf larval eyes  WT  rep3,Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next  Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression  DESeq2 1.34.0 was used in R 4.1.3  using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific  manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 /  adult retina Table S3  we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 /  and WT replicates  derived from 5dpf larval eyes. Genes are ranked by p adj.,eye,,To perform RNA seq on 5 dpf larvae  samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine  anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C  and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed  eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen  then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total  with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,tissue:eye|developmental stage:5 dpf larvae|genotype:WT,GSM7963638,GSM7963638: 5 dpf larval eyes  WT  rep3; Danio rerio; RNA Seq,GSM7963638 r1,GSM7963638,1,To perform RNA seq on 5 dpf larvae  samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine  anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C  and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed  eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen  then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total  with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP476691,,loader:fastq load.py,w6.CCTTGCCGTA-TAGTACACAG.H37F5DSX2_CCTTGCCGTA-TAGTACACAG_L003_R1.fastq.gz w6.CCTTGCCGTA-TAGTACACAG.H37F5DSX2_CCTTGCCGTA-TAGTACACAG_L003_R2.fastq.gz,fastq fastq,11943994904.0,39549652.0,GSM7963638 r1,0:151 1:151,A:3182349655;C:2708994785;G:2976982793;T:3075478468;N:189203,151,151,,,3182349655,2708994785,2976982793,3075478468,189203,SRX22833140,SRS19813578,SRA1765718,"Pathology and Immunology, Washington University School of Medicine","Pathology and Immunology, Washington University School of Medicine",2,0.95522,0.96109,0.04859,0.04955,0.74247,0.74799,0.42424,0.43226,151,151,B,B,biological fallback assumption,illumina,novaseq_era,3prime,random_priming,smarter,bulk,unknown,unknown,,United States,2023-12-08,Larval,Larval,Eye,Sensory System
29111,SRR27151547,SRX22833139,SRS19813579,SRP476691,PRJNA1050282,Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf whole eye],GSE249751,Transcriptome Analysis,The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here  we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 /  retina  red cones are transformed to hybrid red/UV sensitive cones  green cones are transfated to blue cones  and the number of rods is greatly reduced. In the mouse Samd7 /  retina  dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether  Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones  thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf larvae  samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine  anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C  and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed  eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen  then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total  with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces.,parent bioproject:PRJNA1050288,pubmed:39531499,,5 dpf larval eyes  WT  rep2,GSM7963637,,source name:eye|tissue:eye|developmental stage:5 dpf larvae|genotype:WT|geo loc name:missing|collection date:missing,5 dpf larval eyes  WT  rep2,Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next  Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression  DESeq2 1.34.0 was used in R 4.1.3  using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific  manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 /  adult retina Table S3  we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 /  and WT replicates  derived from 5dpf larval eyes. Genes are ranked by p adj.,eye,,To perform RNA seq on 5 dpf larvae  samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine  anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C  and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed  eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen  then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total  with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,tissue:eye|developmental stage:5 dpf larvae|genotype:WT,GSM7963637,GSM7963637: 5 dpf larval eyes  WT  rep2; Danio rerio; RNA Seq,GSM7963637 r1,GSM7963637,1,To perform RNA seq on 5 dpf larvae  samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine  anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C  and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed  eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen  then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total  with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP476691,,loader:fastq load.py,w8.AATCTCAGGC-TCACCAGGAC.H37F5DSX2_AATCTCAGGC-TCACCAGGAC_L003_R1.fastq.gz w8.AATCTCAGGC-TCACCAGGAC.H37F5DSX2_AATCTCAGGC-TCACCAGGAC_L003_R2.fastq.gz,fastq fastq,10471048190.0,34672345.0,GSM7963637 r1,0:151 1:151,A:2767212261;C:2402799397;G:2644524852;T:2656347723;N:163957,151,151,,,2767212261,2402799397,2644524852,2656347723,163957,SRX22833139,SRS19813579,SRA1765718,"Pathology and Immunology, Washington University School of Medicine","Pathology and Immunology, Washington University School of Medicine",2,0.95909,0.96443,0.04132,0.04289,0.74217,0.74631,0.40379,0.41076,151,151,B,B,biological fallback assumption,illumina,novaseq_era,3prime,random_priming,smarter,bulk,unknown,unknown,,United States,2023-12-08,Larval,Larval,Eye,Sensory System
29112,SRR27151548,SRX22833138,SRS19813580,SRP476691,PRJNA1050282,Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf whole eye],GSE249751,Transcriptome Analysis,The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here  we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 /  retina  red cones are transformed to hybrid red/UV sensitive cones  green cones are transfated to blue cones  and the number of rods is greatly reduced. In the mouse Samd7 /  retina  dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether  Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones  thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf larvae  samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine  anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C  and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed  eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen  then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total  with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces.,parent bioproject:PRJNA1050288,pubmed:39531499,,5 dpf larval eyes  WT  rep1,GSM7963636,,source name:eye|tissue:eye|developmental stage:5 dpf larvae|genotype:WT|geo loc name:missing|collection date:missing,5 dpf larval eyes  WT  rep1,Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next  Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression  DESeq2 1.34.0 was used in R 4.1.3  using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific  manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 /  adult retina Table S3  we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 /  and WT replicates  derived from 5dpf larval eyes. Genes are ranked by p adj.,eye,,To perform RNA seq on 5 dpf larvae  samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine  anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C  and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed  eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen  then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total  with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,tissue:eye|developmental stage:5 dpf larvae|genotype:WT,GSM7963636,GSM7963636: 5 dpf larval eyes  WT  rep1; Danio rerio; RNA Seq,GSM7963636 r1,GSM7963636,1,To perform RNA seq on 5 dpf larvae  samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine  anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C  and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed  eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen  then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total  with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP476691,,loader:fastq load.py,w15.AACAGGCAAG-TTCATGGAAG.H37F5DSX2_AACAGGCAAG-TTCATGGAAG_L003_R1.fastq.gz w15.AACAGGCAAG-TTCATGGAAG.H37F5DSX2_AACAGGCAAG-TTCATGGAAG_L003_R2.fastq.gz,fastq fastq,11852131940.0,39245470.0,GSM7963636 r1,0:151 1:151,A:3157263060;C:2691924686;G:2998627934;T:3004129760;N:186500,151,151,,,3157263060,2691924686,2998627934,3004129760,186500,SRX22833138,SRS19813580,SRA1765718,"Pathology and Immunology, Washington University School of Medicine","Pathology and Immunology, Washington University School of Medicine",2,0.95652,0.96043,0.05012,0.05113,0.73198,0.73637,0.42361,0.41636,151,151,B,B,biological fallback assumption,illumina,novaseq_era,3prime,random_priming,smarter,bulk,unknown,unknown,,United States,2023-12-08,Larval,Larval,Eye,Sensory System
29119,SRR27151354,SRX22832956,SRS19813396,SRP476687,PRJNA1050291,Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf td],GSE249755,Transcriptome Analysis,The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here  we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 /  retina  red cones are transformed to hybrid red/UV sensitive cones  green cones are transfated to blue cones  and the number of rods is greatly reduced. In the mouse Samd7 /  retina  dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether  Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones  thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf thrb:tdTomato+ cells  the following crosses were performed: WT × WT;thrb:tdTomato+/  or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS as described in another section. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen  and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total  with RNA integrity RIN scores ranging from 7.4 8.4.,parent bioproject:PRJNA1050288,pubmed:39531499,,thrb:tdTomato+ cells from 5 dpf larval eyes  samd7 /   rep3,GSM7963660,,source name:eye|cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / |geo loc name:missing|collection date:missing,thrb:tdTomato+ cells from 5 dpf larval eyes  samd7 /   rep3,Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next  Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression  DESeq2 1.34.0 was used in R 4.1.3  using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific  manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 /  adult retina Table S3  we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 /  and WT replicates  derived from 5dpf thrb:tdTomato+ cells. Genes are ranked by p adj.,eye,,To perform RNA seq on 5 dpf thrb:tdTomato+ cells  the following crosses were performed: WT × WT;thrb:tdTomato+/  or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen  and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total  with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / ,GSM7963660,GSM7963660: thrb:tdTomato+ cells from 5 dpf larval eyes  samd7 /   rep3; Danio rerio; RNA Seq,GSM7963660 r1,GSM7963660,1,To perform RNA seq on 5 dpf thrb:tdTomato+ cells  the following crosses were performed: WT × WT;thrb:tdTomato+/  or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen  and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total  with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP476687,,loader:fastq load.py,samd7_1_rna_6_2_22.AGAAGGAACA-TGGTAGATGC.HNGJJDSX3_AGAAGGAACA-TGGTAGATGC_L003_R1.fastq.gz samd7_1_rna_6_2_22.AGAAGGAACA-TGGTAGATGC.HNGJJDSX3_AGAAGGAACA-TGGTAGATGC_L003_R2.fastq.gz,fastq fastq,10483631624.0,34714012.0,GSM7963660 r1,0:151 1:151,A:2778161057;C:2394557806;G:2692864342;T:2618022344;N:26075,151,151,,,2778161057,2394557806,2692864342,2618022344,26075,SRX22832956,SRS19813396,SRA1765707,"Pathology and Immunology, Washington University School of Medicine","Pathology and Immunology, Washington University School of Medicine",2,0.91943,0.91641,0.08589,0.08581,0.78748,0.79774,0.5086,0.50263,151,151,B,B,biological fallback assumption,illumina,novaseq_era,3prime,cdna_unspecified,smarter,bulk,unknown,unknown,,United States,2023-12-08,Larval,Larval,Eye,Sensory System
29120,SRR27151355,SRX22832955,SRS19813395,SRP476687,PRJNA1050291,Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf td],GSE249755,Transcriptome Analysis,The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here  we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 /  retina  red cones are transformed to hybrid red/UV sensitive cones  green cones are transfated to blue cones  and the number of rods is greatly reduced. In the mouse Samd7 /  retina  dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether  Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones  thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf thrb:tdTomato+ cells  the following crosses were performed: WT × WT;thrb:tdTomato+/  or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS as described in another section. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen  and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total  with RNA integrity RIN scores ranging from 7.4 8.4.,parent bioproject:PRJNA1050288,pubmed:39531499,,thrb:tdTomato+ cells from 5 dpf larval eyes  samd7 /   rep2,GSM7963659,,source name:eye|cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / |geo loc name:missing|collection date:missing,thrb:tdTomato+ cells from 5 dpf larval eyes  samd7 /   rep2,Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next  Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression  DESeq2 1.34.0 was used in R 4.1.3  using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific  manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 /  adult retina Table S3  we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 /  and WT replicates  derived from 5dpf thrb:tdTomato+ cells. Genes are ranked by p adj.,eye,,To perform RNA seq on 5 dpf thrb:tdTomato+ cells  the following crosses were performed: WT × WT;thrb:tdTomato+/  or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen  and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total  with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / ,GSM7963659,GSM7963659: thrb:tdTomato+ cells from 5 dpf larval eyes  samd7 /   rep2; Danio rerio; RNA Seq,GSM7963659 r1,GSM7963659,1,To perform RNA seq on 5 dpf thrb:tdTomato+ cells  the following crosses were performed: WT × WT;thrb:tdTomato+/  or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen  and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total  with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP476687,,loader:fastq load.py,samd7_1_rna_6_1_22.AGACAGACGT-TATCAGTGCA.HNGJJDSX3_AGACAGACGT-TATCAGTGCA_L003_R1.fastq.gz samd7_1_rna_6_1_22.AGACAGACGT-TATCAGTGCA.HNGJJDSX3_AGACAGACGT-TATCAGTGCA_L003_R2.fastq.gz,fastq fastq,12234446424.0,40511412.0,GSM7963659 r1,0:151 1:151,A:3397427491;C:2650252489;G:2963872812;T:3222862933;N:30699,151,151,,,3397427491,2650252489,2963872812,3222862933,30699,SRX22832955,SRS19813395,SRA1765707,"Pathology and Immunology, Washington University School of Medicine","Pathology and Immunology, Washington University School of Medicine",2,0.89702,0.8967,0.14358,0.14658,0.77309,0.77962,0.5468,0.44645,151,151,B,B,biological fallback assumption,illumina,novaseq_era,3prime,cdna_unspecified,smarter,bulk,unknown,unknown,,United States,2023-12-08,Larval,Larval,Eye,Sensory System
29121,SRR27151356,SRX22832954,SRS19813394,SRP476687,PRJNA1050291,Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf td],GSE249755,Transcriptome Analysis,The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here  we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 /  retina  red cones are transformed to hybrid red/UV sensitive cones  green cones are transfated to blue cones  and the number of rods is greatly reduced. In the mouse Samd7 /  retina  dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether  Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones  thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf thrb:tdTomato+ cells  the following crosses were performed: WT × WT;thrb:tdTomato+/  or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS as described in another section. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen  and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total  with RNA integrity RIN scores ranging from 7.4 8.4.,parent bioproject:PRJNA1050288,pubmed:39531499,,thrb:tdTomato+ cells from 5 dpf larval eyes  samd7 /   rep1,GSM7963658,,source name:eye|cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / |geo loc name:missing|collection date:missing,thrb:tdTomato+ cells from 5 dpf larval eyes  samd7 /   rep1,Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next  Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression  DESeq2 1.34.0 was used in R 4.1.3  using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific  manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 /  adult retina Table S3  we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 /  and WT replicates  derived from 5dpf thrb:tdTomato+ cells. Genes are ranked by p adj.,eye,,To perform RNA seq on 5 dpf thrb:tdTomato+ cells  the following crosses were performed: WT × WT;thrb:tdTomato+/  or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen  and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total  with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / ,GSM7963658,GSM7963658: thrb:tdTomato+ cells from 5 dpf larval eyes  samd7 /   rep1; Danio rerio; RNA Seq,GSM7963658 r1,GSM7963658,1,To perform RNA seq on 5 dpf thrb:tdTomato+ cells  the following crosses were performed: WT × WT;thrb:tdTomato+/  or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen  and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total  with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP476687,,loader:fastq load.py,samd7_rna_5_10_22.CAATTCACGC-ACATCCTGCG.HNGJJDSX3_CAATTCACGC-ACATCCTGCG_L003_R1.fastq.gz samd7_rna_5_10_22.CAATTCACGC-ACATCCTGCG.HNGJJDSX3_CAATTCACGC-ACATCCTGCG_L003_R2.fastq.gz,fastq fastq,11352128660.0,37589830.0,GSM7963658 r1,0:151 1:151,A:3035915425;C:2447970512;G:2970538003;T:2897676299;N:28421,151,151,,,3035915425,2447970512,2970538003,2897676299,28421,SRX22832954,SRS19813394,SRA1765707,"Pathology and Immunology, Washington University School of Medicine","Pathology and Immunology, Washington University School of Medicine",2,0.90706,0.91061,0.09833,0.102,0.78186,0.78948,0.54785,0.47215,151,151,B,B,biological fallback assumption,illumina,novaseq_era,3prime,cdna_unspecified,smarter,bulk,unknown,unknown,,United States,2023-12-08,Larval,Larval,Eye,Sensory System
29122,SRR27151357,SRX22832953,SRS19813393,SRP476687,PRJNA1050291,Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf td],GSE249755,Transcriptome Analysis,The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here  we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 /  retina  red cones are transformed to hybrid red/UV sensitive cones  green cones are transfated to blue cones  and the number of rods is greatly reduced. In the mouse Samd7 /  retina  dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether  Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones  thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf thrb:tdTomato+ cells  the following crosses were performed: WT × WT;thrb:tdTomato+/  or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS as described in another section. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen  and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total  with RNA integrity RIN scores ranging from 7.4 8.4.,parent bioproject:PRJNA1050288,pubmed:39531499,,thrb:tdTomato+ cells from 5 dpf larval eyes  WT  rep3,GSM7963657,,source name:eye|cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:WT|geo loc name:missing|collection date:missing,thrb:tdTomato+ cells from 5 dpf larval eyes  WT  rep3,Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next  Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression  DESeq2 1.34.0 was used in R 4.1.3  using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific  manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 /  adult retina Table S3  we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 /  and WT replicates  derived from 5dpf thrb:tdTomato+ cells. Genes are ranked by p adj.,eye,,To perform RNA seq on 5 dpf thrb:tdTomato+ cells  the following crosses were performed: WT × WT;thrb:tdTomato+/  or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen  and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total  with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:WT,GSM7963657,GSM7963657: thrb:tdTomato+ cells from 5 dpf larval eyes  WT  rep3; Danio rerio; RNA Seq,GSM7963657 r1,GSM7963657,1,To perform RNA seq on 5 dpf thrb:tdTomato+ cells  the following crosses were performed: WT × WT;thrb:tdTomato+/  or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen  and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total  with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP476687,,loader:fastq load.py,wt_3_a_rna_5_12_22.CACCATTGTA-TACCAATTGC.HNGJJDSX3_CACCATTGTA-TACCAATTGC_L003_R1.fastq.gz wt_3_a_rna_5_12_22.CACCATTGTA-TACCAATTGC.HNGJJDSX3_CACCATTGTA-TACCAATTGC_L003_R2.fastq.gz,fastq fastq,9723911364.0,32198382.0,GSM7963657 r1,0:151 1:151,A:2610813507;C:2104688166;G:2520224929;T:2488160661;N:24101,151,151,,,2610813507,2104688166,2520224929,2488160661,24101,SRX22832953,SRS19813393,SRA1765707,"Pathology and Immunology, Washington University School of Medicine","Pathology and Immunology, Washington University School of Medicine",2,0.62594,0.60745,0.07012,0.07039,0.81728,0.82578,0.50983,0.57347,151,151,B,B,biological fallback assumption,illumina,novaseq_era,3prime,cdna_unspecified,smarter,bulk,unknown,unknown,,United States,2023-12-08,Larval,Larval,Eye,Sensory System
29123,SRR27151358,SRX22832952,SRS19813392,SRP476687,PRJNA1050291,Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf td],GSE249755,Transcriptome Analysis,The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here  we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 /  retina  red cones are transformed to hybrid red/UV sensitive cones  green cones are transfated to blue cones  and the number of rods is greatly reduced. In the mouse Samd7 /  retina  dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether  Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones  thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf thrb:tdTomato+ cells  the following crosses were performed: WT × WT;thrb:tdTomato+/  or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS as described in another section. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen  and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total  with RNA integrity RIN scores ranging from 7.4 8.4.,parent bioproject:PRJNA1050288,pubmed:39531499,,thrb:tdTomato+ cells from 5 dpf larval eyes  WT  rep2,GSM7963656,,source name:eye|cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:WT|geo loc name:missing|collection date:missing,thrb:tdTomato+ cells from 5 dpf larval eyes  WT  rep2,Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next  Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression  DESeq2 1.34.0 was used in R 4.1.3  using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific  manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 /  adult retina Table S3  we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 /  and WT replicates  derived from 5dpf thrb:tdTomato+ cells. Genes are ranked by p adj.,eye,,To perform RNA seq on 5 dpf thrb:tdTomato+ cells  the following crosses were performed: WT × WT;thrb:tdTomato+/  or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen  and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total  with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:WT,GSM7963656,GSM7963656: thrb:tdTomato+ cells from 5 dpf larval eyes  WT  rep2; Danio rerio; RNA Seq,GSM7963656 r1,GSM7963656,1,To perform RNA seq on 5 dpf thrb:tdTomato+ cells  the following crosses were performed: WT × WT;thrb:tdTomato+/  or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen  and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total  with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP476687,,loader:fastq load.py,wt_2_b_rna_5_12_22.AAGACTCTCG-TAAGTCTGGC.HNGJJDSX3_AAGACTCTCG-TAAGTCTGGC_L003_R1.fastq.gz wt_2_b_rna_5_12_22.AAGACTCTCG-TAAGTCTGGC.HNGJJDSX3_AAGACTCTCG-TAAGTCTGGC_L003_R2.fastq.gz,fastq fastq,10722667946.0,35505523.0,GSM7963656 r1,0:151 1:151,A:2872256957;C:2358147851;G:2771142656;T:2721093653;N:26829,151,151,,,2872256957,2358147851,2771142656,2721093653,26829,SRX22832952,SRS19813392,SRA1765707,"Pathology and Immunology, Washington University School of Medicine","Pathology and Immunology, Washington University School of Medicine",2,0.90028,0.8986,0.10012,0.10225,0.7973,0.80389,0.56209,0.56007,151,151,B,B,biological fallback assumption,illumina,novaseq_era,3prime,cdna_unspecified,smarter,bulk,unknown,unknown,,United States,2023-12-08,Larval,Larval,Eye,Sensory System
29124,SRR27151359,SRX22832951,SRS19813391,SRP476687,PRJNA1050291,Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf td],GSE249755,Transcriptome Analysis,The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here  we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 /  retina  red cones are transformed to hybrid red/UV sensitive cones  green cones are transfated to blue cones  and the number of rods is greatly reduced. In the mouse Samd7 /  retina  dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether  Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones  thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf thrb:tdTomato+ cells  the following crosses were performed: WT × WT;thrb:tdTomato+/  or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS as described in another section. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen  and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total  with RNA integrity RIN scores ranging from 7.4 8.4.,parent bioproject:PRJNA1050288,pubmed:39531499,,thrb:tdTomato+ cells from 5 dpf larval eyes  WT  rep1,GSM7963655,,source name:eye|cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:WT|geo loc name:missing|collection date:missing,thrb:tdTomato+ cells from 5 dpf larval eyes  WT  rep1,Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next  Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression  DESeq2 1.34.0 was used in R 4.1.3  using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific  manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 /  adult retina Table S3  we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 /  and WT replicates  derived from 5dpf thrb:tdTomato+ cells. Genes are ranked by p adj.,eye,,To perform RNA seq on 5 dpf thrb:tdTomato+ cells  the following crosses were performed: WT × WT;thrb:tdTomato+/  or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen  and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total  with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:WT,GSM7963655,GSM7963655: thrb:tdTomato+ cells from 5 dpf larval eyes  WT  rep1; Danio rerio; RNA Seq,GSM7963655 r1,GSM7963655,1,To perform RNA seq on 5 dpf thrb:tdTomato+ cells  the following crosses were performed: WT × WT;thrb:tdTomato+/  or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen  and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total  with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples  and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18  duty factor 20%  cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase  Klenow Fragment DNA Polymerase  and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo   and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples  and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags  using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP476687,,loader:fastq load.py,wt_rna_5_10_22.CCACCTTCAA-ACCATCGAGG.HNGJJDSX3_CCACCTTCAA-ACCATCGAGG_L003_R1.fastq.gz wt_rna_5_10_22.CCACCTTCAA-ACCATCGAGG.HNGJJDSX3_CCACCTTCAA-ACCATCGAGG_L003_R2.fastq.gz,fastq fastq,9875387618.0,32699959.0,GSM7963655 r1,0:151 1:151,A:2640364023;C:2083842037;G:2621465276;T:2529691405;N:24877,151,151,,,2640364023,2083842037,2621465276,2529691405,24877,SRX22832951,SRS19813391,SRA1765707,"Pathology and Immunology, Washington University School of Medicine","Pathology and Immunology, Washington University School of Medicine",2,0.90131,0.90429,0.10837,0.1106,0.79523,0.80182,0.50934,0.58039,151,151,B,B,biological fallback assumption,illumina,novaseq_era,3prime,cdna_unspecified,smarter,bulk,unknown,unknown,,United States,2023-12-08,Larval,Larval,Eye,Sensory System
29174,SRR27237224,SRX22915658,SRS19883761,SRP478464,PRJNA1053781,Single cell gene expression profie of developing photoreceptor cells in larval zebrafish,GSE250379,Other,Molecular underpinnings of vertebrate retinal differentiation and maturation are poorly understood  particularly for non mammalian species. We generated single cell transcriptome data from the larval zebrafish retina and characterized gene expression diversity among photoreceptor subtypes and their progenitors. Overall design: GFP positive differentiating photoreceptor cells and bipolar cells were collected from 4 dpf larval transgenic zebrafish  Tgcrx:EGFPstl887  using fluorescence activated cell sorting.,parent bioproject:PRJNA1050288,pubmed:39531499,,retina  scRNA seq,GSM7978132,,source name:retina|tissue:retina|genotype:Tgcrx:EGFPstl887|developmental stage:4 dpf|geo loc name:missing|collection date:missing,retina  scRNA seq,Read alignment and initial quality control were performed using Cell Ranger software version 7.0.0  10X Genomics. Assembly: GRCz11 Supplementary files format and content: Tab separated value file and matrix file,retina,,Fifty heads were dissected from 4 dpf heterozygous Tgcrx:GFPstl887Tg larvae. Following dissection  eyes were stored in ice cold Hanks’ Balanced Salt Solution HBSS until all eyes were harvested. Once the eyes were collected  HBSS was removed and the eyes were incubated in 400 µl of calcium/magnesium free HBSS containing 0.4 mg papain Worthington Biochem for 15 min at 37°C. 800 µl of 10% fetal bovine serum FBS in Dulbecco's Modified Eagle Medium DMEM containing 5mM MgCl2 and 120 units DNaseI Roche were added to the mixture and incubated for 5 min at 37°C. Cells were then resuspended in 300 µl of sorting buffer 2.5 mM EDTA  25 mM HEPES  1% bovine serum albumin BSA in calcium/magnesium free HBSS. Cells were sorted on an Aria II FACS machine BD biosciences with gating based on forward scatter  side scatter  and GFP fluorescence and collected in 700 μl of D PBS without xxx+ and Mg2+  supplemented with 0.4 % BSA D PBS CMF in 1.5 ml microcentrifuge tubes. The collected cells were then centrifuged at 300×g for 5 min  washed with D PBS CMF  centrifuged  and supernatant reduced to 80 µl. Cell density was quantified on a hemocytometer  and  5000 cells were used for single cell library preparation. A library for single cell RNA seq was constructed with the Chromium v3  platform  10X  Genomics   Pleasanton   CA according to the manufacturer protocol.,,tissue:retina|genotype:Tgcrx:EGFPstl887|developmental stage:4 dpf,GSM7978132,GSM7978132: retina  scRNA seq; Danio rerio; RNA Seq,GSM7978132 r1,GSM7978132,1,Fifty heads were dissected from 4 dpf heterozygous Tgcrx:GFPstl887Tg larvae. Following dissection  eyes were stored in ice cold Hanks' Balanced Salt Solution HBSS until all eyes were harvested. Once the eyes were collected  HBSS was removed and the eyes were incubated in 400 µl of calcium/magnesium free HBSS containing 0.4 mg papain Worthington Biochem for 15 min at 37°C. 800 µl of 10% fetal bovine serum FBS in Dulbecco's Modified Eagle Medium DMEM containing 5mM MgCl2 and 120 units DNaseI Roche were added to the mixture and incubated for 5 min at 37°C. Cells were then resuspended in 300 µl of sorting buffer 2.5 mM EDTA  25 mM HEPES  1% bovine serum albumin BSA in calcium/magnesium free HBSS. Cells were sorted on an Aria II FACS machine BD biosciences with gating based on forward scatter  side scatter  and GFP fluorescence and collected in 700 μl of D PBS without xxx+ and Mg2+  supplemented with 0.4 % BSA D PBS CMF in 1.5 ml microcentrifuge tubes. The collected cells were then centrifuged at 300×g for 5 min  washed with D PBS CMF  centrifuged  and supernatant reduced to 80 µl. Cell density was quantified on a hemocytometer  and  5000 cells were used for single cell library preparation. A library for single cell RNA seq was constructed with the Chromium v3  platform  10X  Genomics   Pleasanton   CA according to the manufacturer protocol.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP478464,,loader:fastq load.py,crx.crx_S1_L003_R1_001.fastq.gz crx.crx_S1_L003_R2_001.fastq.gz,fastq fastq,45170138494.0,253764823.0,GSM7978132 r1,0:28 1:150,A:13826023801;C:9023674621;G:9590649474;T:12728940802;N:849796,28,150,,,13826023801,9023674621,9590649474,12728940802,849796,SRX22915658,SRS19883761,SRA1770358,"Pathology and Immunology, Washington University School of Medicine","Pathology and Immunology, Washington University School of Medicine",2,0.0046,0.87155,0.00206,0.2263,0.99168,0.77784,0.30223,0.50112,28,150,T,B,sc-like readlen,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-12-17,Larval,Larval,Eye,Sensory System
31849,SRR28735527,SRX24301508,SRS21065427,SRP502615,PRJNA1101945,Loss of Stim2 in zebrafish induces retinal gene expression cellular changes resembling glaucoma characteristics [RNA seq],GSE264309,Transcriptome Analysis,Calcium is involved in vision processes in retina and is implicated in various pathologies including glaucoma. Rods are protected against prolonged lowering of intracellular calcium ion concentrations by Store Operated Calcium Entry SOCE. We showed zebrafish lacking SOCE calcium sensor Stim2 had problem with vision as indicated by behavior tests  staining of retina and downregulation of genes related to light perception. In this work we aimed to understand the mechanism responsible for the vision problems in stim2 zebrafish knockout. scRNA sequencing of neuronal origin cells from brains of 5 dpf larvae identified 27 clusters. Differently expressed genes were detected in ten clusters including amacrine and GABAergic retinal interneurons and GABAergic optic tectum cells. In five clusters the proportion of cells in stim2 KO fish versus control was significantly decreased including GABAergic diencephalon and optic tectum  and was increased in amacrine and GABAergic retinal interneurons. Transmission Electron Microscopy in stim2 KO fish revealed decrease in width of inner plexiform layer IPL   ganglion cells and their dendrites numbers what is characteristic for glaucoma. Analysis of cell density in the inner nucleus layer  which includes amacrine cells among others  showed a significant decrease in the number of GABAergic neurons. The area of cristae in photoreceptor mitochondria was statistically lower in stim2 KO than in control retinas. Overall design: AB and double knockout for stim2   stim2a;stim2b /  zebrafish 5dpf larvae were used to isolate the total RNA from their eyes. The experimental setup involved 3 replicates per each condition stim2 KO vs control consisting of 35 eyes per repetition.,parent bioproject:PRJNA1101942,pubmed:39424970,,stim2KO  repllicate 3  RNASeq,GSM8215878,,source name:eye|tissue:eye|genotype:stim2a;stim2b / |age:5 dpf|geo loc name:missing|collection date:missing,stim2KO  repllicate 3  RNASeq,Raw RNA seq data were converted to FASTQ format with bcl2fastq2 v2.17  Illumina  and FastQC v0.11.4 was used to assess the quality of each sequencing run. We then utilized the SQuIRE pipeline45 for subsequent steps. STAR version 2.5.3a  StringTie version 1.3.3b  and DESeq2 version 1.16.149 were used within the SQuIRE pipeline for read alignment  transcript assembly  and quantification for each replicate  and differential gene expression analysis  respectively. Assembly: Danio rerio GRCz11 Supplementary files format and content: Tab separated files with raw reads for each sample,eye,,Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260  280  and 230 nm  and Bioanalyzer Nano RNA chip Agilent  USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research  Irvine  CA  USA according to the manufacturer’s recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.,,tissue:eye|genotype:stim2a;stim2b / |age:5 dpf,GSM8215878,GSM8215878: stim2KO  repllicate 3  RNASeq; Danio rerio; RNA Seq,GSM8215878 r1,GSM8215878,1,Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260  280  and 230 nm  and Bioanalyzer Nano RNA chip Agilent  USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research  Irvine  CA  USA according to the manufacturer's recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP502615,,loader:fastq load.py,mut_rep_3_R1_001.fastq.gz mut_rep_3_R2_001.fastq.gz,fastq fastq,5198552618.0,25735409.0,GSM8215878 r1,0:101 1:101,A:1302884238;C:1298420335;G:1334893927;T:1262296331;N:57787,101,101,,,1302884238,1298420335,1334893927,1262296331,57787,SRX24301508,SRS21065427,SRA1848589,"Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS","Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS",2,0.95719,0.95856,0.05756,0.05712,0.70534,0.70851,0.42046,0.41786,101,101,B,B,biological fallback assumption,illumina,nextseq,unknown,cdna_unspecified,trueseq,sc_generic,single_cell_generic,generic-scrnaseq-only,,Poland,2024-04-18,Larval,Larval,Eye,Sensory System
31850,SRR28735528,SRX24301507,SRS21065426,SRP502615,PRJNA1101945,Loss of Stim2 in zebrafish induces retinal gene expression cellular changes resembling glaucoma characteristics [RNA seq],GSE264309,Transcriptome Analysis,Calcium is involved in vision processes in retina and is implicated in various pathologies including glaucoma. Rods are protected against prolonged lowering of intracellular calcium ion concentrations by Store Operated Calcium Entry SOCE. We showed zebrafish lacking SOCE calcium sensor Stim2 had problem with vision as indicated by behavior tests  staining of retina and downregulation of genes related to light perception. In this work we aimed to understand the mechanism responsible for the vision problems in stim2 zebrafish knockout. scRNA sequencing of neuronal origin cells from brains of 5 dpf larvae identified 27 clusters. Differently expressed genes were detected in ten clusters including amacrine and GABAergic retinal interneurons and GABAergic optic tectum cells. In five clusters the proportion of cells in stim2 KO fish versus control was significantly decreased including GABAergic diencephalon and optic tectum  and was increased in amacrine and GABAergic retinal interneurons. Transmission Electron Microscopy in stim2 KO fish revealed decrease in width of inner plexiform layer IPL   ganglion cells and their dendrites numbers what is characteristic for glaucoma. Analysis of cell density in the inner nucleus layer  which includes amacrine cells among others  showed a significant decrease in the number of GABAergic neurons. The area of cristae in photoreceptor mitochondria was statistically lower in stim2 KO than in control retinas. Overall design: AB and double knockout for stim2   stim2a;stim2b /  zebrafish 5dpf larvae were used to isolate the total RNA from their eyes. The experimental setup involved 3 replicates per each condition stim2 KO vs control consisting of 35 eyes per repetition.,parent bioproject:PRJNA1101942,pubmed:39424970,,stim2KO  repllicate 2  RNASeq,GSM8215877,,source name:eye|tissue:eye|genotype:stim2a;stim2b / |age:5 dpf|geo loc name:missing|collection date:missing,stim2KO  repllicate 2  RNASeq,Raw RNA seq data were converted to FASTQ format with bcl2fastq2 v2.17  Illumina  and FastQC v0.11.4 was used to assess the quality of each sequencing run. We then utilized the SQuIRE pipeline45 for subsequent steps. STAR version 2.5.3a  StringTie version 1.3.3b  and DESeq2 version 1.16.149 were used within the SQuIRE pipeline for read alignment  transcript assembly  and quantification for each replicate  and differential gene expression analysis  respectively. Assembly: Danio rerio GRCz11 Supplementary files format and content: Tab separated files with raw reads for each sample,eye,,Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260  280  and 230 nm  and Bioanalyzer Nano RNA chip Agilent  USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research  Irvine  CA  USA according to the manufacturer’s recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.,,tissue:eye|genotype:stim2a;stim2b / |age:5 dpf,GSM8215877,GSM8215877: stim2KO  repllicate 2  RNASeq; Danio rerio; RNA Seq,GSM8215877 r1,GSM8215877,1,Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260  280  and 230 nm  and Bioanalyzer Nano RNA chip Agilent  USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research  Irvine  CA  USA according to the manufacturer's recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP502615,,loader:fastq load.py,mut_rep_2_R1_001.fastq.gz mut_rep_2_R2_001.fastq.gz,fastq fastq,5641008570.0,27925785.0,GSM8215877 r1,0:101 1:101,A:1421655053;C:1399106135;G:1439454285;T:1380731092;N:62005,101,101,,,1421655053,1399106135,1439454285,1380731092,62005,SRX24301507,SRS21065426,SRA1848589,"Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS","Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS",2,0.95794,0.9601,0.05457,0.05365,0.70942,0.71151,0.42408,0.42666,101,101,B,B,biological fallback assumption,illumina,nextseq,unknown,cdna_unspecified,trueseq,sc_generic,single_cell_generic,generic-scrnaseq-only,,Poland,2024-04-18,Larval,Larval,Eye,Sensory System
31851,SRR28735529,SRX24301506,SRS21065425,SRP502615,PRJNA1101945,Loss of Stim2 in zebrafish induces retinal gene expression cellular changes resembling glaucoma characteristics [RNA seq],GSE264309,Transcriptome Analysis,Calcium is involved in vision processes in retina and is implicated in various pathologies including glaucoma. Rods are protected against prolonged lowering of intracellular calcium ion concentrations by Store Operated Calcium Entry SOCE. We showed zebrafish lacking SOCE calcium sensor Stim2 had problem with vision as indicated by behavior tests  staining of retina and downregulation of genes related to light perception. In this work we aimed to understand the mechanism responsible for the vision problems in stim2 zebrafish knockout. scRNA sequencing of neuronal origin cells from brains of 5 dpf larvae identified 27 clusters. Differently expressed genes were detected in ten clusters including amacrine and GABAergic retinal interneurons and GABAergic optic tectum cells. In five clusters the proportion of cells in stim2 KO fish versus control was significantly decreased including GABAergic diencephalon and optic tectum  and was increased in amacrine and GABAergic retinal interneurons. Transmission Electron Microscopy in stim2 KO fish revealed decrease in width of inner plexiform layer IPL   ganglion cells and their dendrites numbers what is characteristic for glaucoma. Analysis of cell density in the inner nucleus layer  which includes amacrine cells among others  showed a significant decrease in the number of GABAergic neurons. The area of cristae in photoreceptor mitochondria was statistically lower in stim2 KO than in control retinas. Overall design: AB and double knockout for stim2   stim2a;stim2b /  zebrafish 5dpf larvae were used to isolate the total RNA from their eyes. The experimental setup involved 3 replicates per each condition stim2 KO vs control consisting of 35 eyes per repetition.,parent bioproject:PRJNA1101942,pubmed:39424970,,stim2KO  repllicate 1  RNASeq,GSM8215876,,source name:eye|tissue:eye|genotype:stim2a;stim2b / |age:5 dpf|geo loc name:missing|collection date:missing,stim2KO  repllicate 1  RNASeq,Raw RNA seq data were converted to FASTQ format with bcl2fastq2 v2.17  Illumina  and FastQC v0.11.4 was used to assess the quality of each sequencing run. We then utilized the SQuIRE pipeline45 for subsequent steps. STAR version 2.5.3a  StringTie version 1.3.3b  and DESeq2 version 1.16.149 were used within the SQuIRE pipeline for read alignment  transcript assembly  and quantification for each replicate  and differential gene expression analysis  respectively. Assembly: Danio rerio GRCz11 Supplementary files format and content: Tab separated files with raw reads for each sample,eye,,Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260  280  and 230 nm  and Bioanalyzer Nano RNA chip Agilent  USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research  Irvine  CA  USA according to the manufacturer’s recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.,,tissue:eye|genotype:stim2a;stim2b / |age:5 dpf,GSM8215876,GSM8215876: stim2KO  repllicate 1  RNASeq; Danio rerio; RNA Seq,GSM8215876 r1,GSM8215876,1,Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260  280  and 230 nm  and Bioanalyzer Nano RNA chip Agilent  USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research  Irvine  CA  USA according to the manufacturer's recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP502615,,loader:fastq load.py,mut_rep_1_R1_001.fastq.gz mut_rep_1_R2_001.fastq.gz,fastq fastq,5147003026.0,25480213.0,GSM8215876 r1,0:101 1:101,A:1302966446;C:1274828722;G:1325578656;T:1243571921;N:57281,101,101,,,1302966446,1274828722,1325578656,1243571921,57281,SRX24301506,SRS21065425,SRA1848589,"Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS","Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS",2,0.95963,0.96036,0.05042,0.05053,0.71275,0.71725,0.41458,0.41703,101,101,B,B,biological fallback assumption,illumina,nextseq,unknown,cdna_unspecified,trueseq,sc_generic,single_cell_generic,generic-scrnaseq-only,,Poland,2024-04-18,Larval,Larval,Eye,Sensory System
31852,SRR28735530,SRX24301505,SRS21065424,SRP502615,PRJNA1101945,Loss of Stim2 in zebrafish induces retinal gene expression cellular changes resembling glaucoma characteristics [RNA seq],GSE264309,Transcriptome Analysis,Calcium is involved in vision processes in retina and is implicated in various pathologies including glaucoma. Rods are protected against prolonged lowering of intracellular calcium ion concentrations by Store Operated Calcium Entry SOCE. We showed zebrafish lacking SOCE calcium sensor Stim2 had problem with vision as indicated by behavior tests  staining of retina and downregulation of genes related to light perception. In this work we aimed to understand the mechanism responsible for the vision problems in stim2 zebrafish knockout. scRNA sequencing of neuronal origin cells from brains of 5 dpf larvae identified 27 clusters. Differently expressed genes were detected in ten clusters including amacrine and GABAergic retinal interneurons and GABAergic optic tectum cells. In five clusters the proportion of cells in stim2 KO fish versus control was significantly decreased including GABAergic diencephalon and optic tectum  and was increased in amacrine and GABAergic retinal interneurons. Transmission Electron Microscopy in stim2 KO fish revealed decrease in width of inner plexiform layer IPL   ganglion cells and their dendrites numbers what is characteristic for glaucoma. Analysis of cell density in the inner nucleus layer  which includes amacrine cells among others  showed a significant decrease in the number of GABAergic neurons. The area of cristae in photoreceptor mitochondria was statistically lower in stim2 KO than in control retinas. Overall design: AB and double knockout for stim2   stim2a;stim2b /  zebrafish 5dpf larvae were used to isolate the total RNA from their eyes. The experimental setup involved 3 replicates per each condition stim2 KO vs control consisting of 35 eyes per repetition.,parent bioproject:PRJNA1101942,pubmed:39424970,,Tg  repllicate 3  RNASeq,GSM8215875,,source name:eye|tissue:eye|genotype:AB|age:5 dpf|geo loc name:missing|collection date:missing,Tg  repllicate 3  RNASeq,Raw RNA seq data were converted to FASTQ format with bcl2fastq2 v2.17  Illumina  and FastQC v0.11.4 was used to assess the quality of each sequencing run. We then utilized the SQuIRE pipeline45 for subsequent steps. STAR version 2.5.3a  StringTie version 1.3.3b  and DESeq2 version 1.16.149 were used within the SQuIRE pipeline for read alignment  transcript assembly  and quantification for each replicate  and differential gene expression analysis  respectively. Assembly: Danio rerio GRCz11 Supplementary files format and content: Tab separated files with raw reads for each sample,eye,,Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260  280  and 230 nm  and Bioanalyzer Nano RNA chip Agilent  USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research  Irvine  CA  USA according to the manufacturer’s recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.,,tissue:eye|genotype:AB|age:5 dpf,GSM8215875,GSM8215875: Tg  repllicate 3  RNASeq; Danio rerio; RNA Seq,GSM8215875 r1,GSM8215875,1,Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260  280  and 230 nm  and Bioanalyzer Nano RNA chip Agilent  USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research  Irvine  CA  USA according to the manufacturer's recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP502615,,loader:fastq load.py,wt_rep_3_R1_001.fastq.gz wt_rep_3_R2_001.fastq.gz,fastq fastq,4648345422.0,23011611.0,GSM8215875 r1,0:101 1:101,A:1186641846;C:1144216110;G:1192801277;T:1124633797;N:52392,101,101,,,1186641846,1144216110,1192801277,1124633797,52392,SRX24301505,SRS21065424,SRA1848589,"Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS","Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS",2,0.9575,0.95756,0.05359,0.05311,0.69875,0.70315,0.42041,0.41054,101,101,B,B,biological fallback assumption,illumina,nextseq,unknown,cdna_unspecified,trueseq,sc_generic,single_cell_generic,generic-scrnaseq-only,,Poland,2024-04-18,Larval,Larval,Eye,Sensory System
31853,SRR28735531,SRX24301504,SRS21065423,SRP502615,PRJNA1101945,Loss of Stim2 in zebrafish induces retinal gene expression cellular changes resembling glaucoma characteristics [RNA seq],GSE264309,Transcriptome Analysis,Calcium is involved in vision processes in retina and is implicated in various pathologies including glaucoma. Rods are protected against prolonged lowering of intracellular calcium ion concentrations by Store Operated Calcium Entry SOCE. We showed zebrafish lacking SOCE calcium sensor Stim2 had problem with vision as indicated by behavior tests  staining of retina and downregulation of genes related to light perception. In this work we aimed to understand the mechanism responsible for the vision problems in stim2 zebrafish knockout. scRNA sequencing of neuronal origin cells from brains of 5 dpf larvae identified 27 clusters. Differently expressed genes were detected in ten clusters including amacrine and GABAergic retinal interneurons and GABAergic optic tectum cells. In five clusters the proportion of cells in stim2 KO fish versus control was significantly decreased including GABAergic diencephalon and optic tectum  and was increased in amacrine and GABAergic retinal interneurons. Transmission Electron Microscopy in stim2 KO fish revealed decrease in width of inner plexiform layer IPL   ganglion cells and their dendrites numbers what is characteristic for glaucoma. Analysis of cell density in the inner nucleus layer  which includes amacrine cells among others  showed a significant decrease in the number of GABAergic neurons. The area of cristae in photoreceptor mitochondria was statistically lower in stim2 KO than in control retinas. Overall design: AB and double knockout for stim2   stim2a;stim2b /  zebrafish 5dpf larvae were used to isolate the total RNA from their eyes. The experimental setup involved 3 replicates per each condition stim2 KO vs control consisting of 35 eyes per repetition.,parent bioproject:PRJNA1101942,pubmed:39424970,,Tg  repllicate 2  RNASeq,GSM8215874,,source name:eye|tissue:eye|genotype:AB|age:5 dpf|geo loc name:missing|collection date:missing,Tg  repllicate 2  RNASeq,Raw RNA seq data were converted to FASTQ format with bcl2fastq2 v2.17  Illumina  and FastQC v0.11.4 was used to assess the quality of each sequencing run. We then utilized the SQuIRE pipeline45 for subsequent steps. STAR version 2.5.3a  StringTie version 1.3.3b  and DESeq2 version 1.16.149 were used within the SQuIRE pipeline for read alignment  transcript assembly  and quantification for each replicate  and differential gene expression analysis  respectively. Assembly: Danio rerio GRCz11 Supplementary files format and content: Tab separated files with raw reads for each sample,eye,,Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260  280  and 230 nm  and Bioanalyzer Nano RNA chip Agilent  USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research  Irvine  CA  USA according to the manufacturer’s recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.,,tissue:eye|genotype:AB|age:5 dpf,GSM8215874,GSM8215874: Tg  repllicate 2  RNASeq; Danio rerio; RNA Seq,GSM8215874 r1,GSM8215874,1,Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260  280  and 230 nm  and Bioanalyzer Nano RNA chip Agilent  USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research  Irvine  CA  USA according to the manufacturer's recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP502615,,loader:fastq load.py,wt_rep_2_R1_001.fastq.gz wt_rep_2_R2_001.fastq.gz,fastq fastq,4769618546.0,23611973.0,GSM8215874 r1,0:101 1:101,A:1218890346;C:1171831021;G:1228410692;T:1150434539;N:51948,101,101,,,1218890346,1171831021,1228410692,1150434539,51948,SRX24301504,SRS21065423,SRA1848589,"Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS","Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS",2,0.95682,0.95517,0.05192,0.05059,0.70849,0.71543,0.41181,0.41549,101,101,B,B,biological fallback assumption,illumina,nextseq,unknown,cdna_unspecified,trueseq,sc_generic,single_cell_generic,generic-scrnaseq-only,,Poland,2024-04-18,Larval,Larval,Eye,Sensory System
31854,SRR28735532,SRX24301503,SRS21065422,SRP502615,PRJNA1101945,Loss of Stim2 in zebrafish induces retinal gene expression cellular changes resembling glaucoma characteristics [RNA seq],GSE264309,Transcriptome Analysis,Calcium is involved in vision processes in retina and is implicated in various pathologies including glaucoma. Rods are protected against prolonged lowering of intracellular calcium ion concentrations by Store Operated Calcium Entry SOCE. We showed zebrafish lacking SOCE calcium sensor Stim2 had problem with vision as indicated by behavior tests  staining of retina and downregulation of genes related to light perception. In this work we aimed to understand the mechanism responsible for the vision problems in stim2 zebrafish knockout. scRNA sequencing of neuronal origin cells from brains of 5 dpf larvae identified 27 clusters. Differently expressed genes were detected in ten clusters including amacrine and GABAergic retinal interneurons and GABAergic optic tectum cells. In five clusters the proportion of cells in stim2 KO fish versus control was significantly decreased including GABAergic diencephalon and optic tectum  and was increased in amacrine and GABAergic retinal interneurons. Transmission Electron Microscopy in stim2 KO fish revealed decrease in width of inner plexiform layer IPL   ganglion cells and their dendrites numbers what is characteristic for glaucoma. Analysis of cell density in the inner nucleus layer  which includes amacrine cells among others  showed a significant decrease in the number of GABAergic neurons. The area of cristae in photoreceptor mitochondria was statistically lower in stim2 KO than in control retinas. Overall design: AB and double knockout for stim2   stim2a;stim2b /  zebrafish 5dpf larvae were used to isolate the total RNA from their eyes. The experimental setup involved 3 replicates per each condition stim2 KO vs control consisting of 35 eyes per repetition.,parent bioproject:PRJNA1101942,pubmed:39424970,,Tg  repllicate 1  RNASeq,GSM8215873,,source name:eye|tissue:eye|genotype:AB|age:5 dpf|geo loc name:missing|collection date:missing,Tg  repllicate 1  RNASeq,Raw RNA seq data were converted to FASTQ format with bcl2fastq2 v2.17  Illumina  and FastQC v0.11.4 was used to assess the quality of each sequencing run. We then utilized the SQuIRE pipeline45 for subsequent steps. STAR version 2.5.3a  StringTie version 1.3.3b  and DESeq2 version 1.16.149 were used within the SQuIRE pipeline for read alignment  transcript assembly  and quantification for each replicate  and differential gene expression analysis  respectively. Assembly: Danio rerio GRCz11 Supplementary files format and content: Tab separated files with raw reads for each sample,eye,,Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260  280  and 230 nm  and Bioanalyzer Nano RNA chip Agilent  USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research  Irvine  CA  USA according to the manufacturer’s recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.,,tissue:eye|genotype:AB|age:5 dpf,GSM8215873,GSM8215873: Tg  repllicate 1  RNASeq; Danio rerio; RNA Seq,GSM8215873 r1,GSM8215873,1,Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260  280  and 230 nm  and Bioanalyzer Nano RNA chip Agilent  USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research  Irvine  CA  USA according to the manufacturer's recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP502615,,loader:fastq load.py,wt_rep_1_R1_001.fastq.gz wt_rep_1_R2_001.fastq.gz,fastq fastq,4771981138.0,23623669.0,GSM8215873 r1,0:101 1:101,A:1222918015;C:1171619846;G:1231393968;T:1145997219;N:52090,101,101,,,1222918015,1171619846,1231393968,1145997219,52090,SRX24301503,SRS21065422,SRA1848589,"Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS","Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS",2,0.95739,0.95621,0.05362,0.0539,0.71301,0.71979,0.42337,0.40836,101,101,B,B,biological fallback assumption,illumina,nextseq,unknown,cdna_unspecified,trueseq,sc_generic,single_cell_generic,generic-scrnaseq-only,,Poland,2024-04-18,Larval,Larval,Eye,Sensory System
32366,SRR29152187,SRX24673329,SRS21408057,SRP509548,PRJNA1115803,Stem and progenitor cell proliferation are independently regulated by cell type specific cyclinD genes,GSE268255,Transcriptome Analysis,Regeneration and homeostatic turnover of solid tissues depend on the proliferation of symmetrically dividing adult stem cells  which either remain stem cells or differentiate based on their niche position. Here we demonstrate that in zebrafish lateral line sensory organs  stem and progenitor cell proliferation are independently regulated by two cyclinD genes. Loss of ccnd2a impairs stem cell proliferation during development  while loss of ccndx disrupts hair cell progenitor proliferation but allows normal differentiation. Notably  ccnd2a can functionally replace ccndx  indicating that the respective effects of these Cyclins on proliferation are due to cell type specific expression. However  even though hair cell progenitors differentiate normally in ccndx mutants  they are mispolarized due to hes2 and Emx2 downregulation. Thus  regulated proliferation ensures that equal numbers of hair cells are polarized in opposite directions. Our study reveals cell type specific roles for cyclinD genes in regulating the different populations of symmetrically dividing cells governing organ development and regeneration  with implications for regenerative medicine and disease. Overall design: Two replicates per sibling or ccndx mutant were used,,,,ccndx sibling2,GSM8288756,,tissue:5dpf zebrafish FACS sorted lateral line cells|cell type:Homostasis|genotype:Wildtype|geo loc name:missing|collection date:missing,ccndx sibling2,Raw sequencing data were processed using 10X Genomics Cell Ranger pipeline. Reads were demultiplexed into Fastq file format using cellranger mkfastq. Genome index was built by cellranger mkref using zebrafish genome danRer11  ensembl 98 gene model. Data were aligned by STAR aligner and cell counts tables were generated using cellranger count function with default parameters. Assembly: danRer11 Supplementary files format and content: single cell  count matrix in hdf5 format  raw data in bam format.,5dpf zebrafish FACS sorted lateral line cells,All cells were collected during homeostasis  no treatments were performed.,5 dpf zebrafish were anesthetized with Tricane then split between two 70mm cell strainers Falcon and placed in 0.25% Trypsin EDTA Gibco on ice. The larvae from each strainer are then added to a 5ml round bottom tube Falcon and the tissue is broken up via slowly pipetting up and down using a pipette man and a glass pipette VWR keeping the tubes on ice. The total time from placing larvae in trypsin to finishing pipetting is approximately 8 minutes. The tissue is then filtered through 70mm filter BD Biosciences  pelleted for 5 min at 2000rpm at 4oC and then the pellet is resuspended in 1xDPBS/0.04%BSA. The pelleting and resuspension in 1xDPBS/0.04%BSA is repeated two more times. post the final pellet the cells are resuspended in 0.5mL 1xDPBS/0.04%BSA then cells are combined and put through another 70mm filter into a 5ml round bottom tube. Cells are then incubated in Draq5 1/200 dilution for 5 minutes on ice. Draq5+/mCherry+ cells were sorted by the Stowers Institute Cytometry Core Facility using a BD Influx Cell Sorter BD Biosciences  San Jose  CA. USA  with cells collected in tubes containing 10mL 1XDPBS/0.04% BSA. Cells were not fixed in methanol as in previous protocols as we found this gave inconsistent results with 10X Genomics version 3 kits. Sorted cells are then spun down at 350rcf at 4oC for 2 min in a swinging bucket centrifuge with slow deceleration. Most of the supernatant is removed and the cells are resuspended in approximately remaining 100mL. Cells deemed to be at least 80% viable were loaded based on live cell concentration onto a Chromium X Series instrument 10x Genomics and libraries prepared using the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 10x Genomics according to manufacturer’s directions. Resulting cDNA and short fragment libraries were checked for quality and quantity using a 2100 Bioanalyzer Agilent Technologies and Qubit Fluorometer Thermo Fisher Scientific. With cells captured estimated at 6 800 9 600 cells per sample  libraries were pooled and sequenced to a depth necessary to achieve at least 30 000 mean reads per cell on an Illumina NextSeq 2000 instrument utilizing RTA and instrument software versions current at the time of processing with the following paired read lengths: 28*10*10*90bp. scRNA seq 10x Genomics,5dpf zebrafish FACS sorted lateral line cells were used. Standard growing condition were used.,cell type:Homostasis|genotype:Wildtype,GSM8288756,GSM8288756: ccndx sibling2; Danio rerio; RNA Seq,GSM8288756 r1,GSM8288756,1,5 dpf zebrafish were anesthetized with Tricane then split between two 70mm cell strainers Falcon and placed in 0.25% Trypsin EDTA Gibco on ice. The larvae from each strainer are then added to a 5ml round bottom tube Falcon and the tissue is broken up via slowly pipetting up and down using a pipette man and a glass pipette VWR keeping the tubes on ice. The total time from placing larvae in trypsin to finishing pipetting is approximately 8 minutes. The tissue is then filtered through 70mm filter BD Biosciences  pelleted for 5 min at 2000rpm at 4oC and then the pellet is resuspended in 1xDPBS/0.04%BSA. The pelleting and resuspension in 1xDPBS/0.04%BSA is repeated two more times. post the final pellet the cells are resuspended in 0.5mL 1xDPBS/0.04%BSA then cells are combined and put through another 70mm filter into a 5ml round bottom tube. Cells are then incubated in Draq5 1/200 dilution for 5 minutes on ice. Draq5+/mCherry+ cells were sorted by the Stowers Institute Cytometry Core Facility using a BD Influx Cell Sorter BD Biosciences  San Jose  CA. USA  with cells collected in tubes containing 10mL 1XDPBS/0.04% BSA. Cells were not fixed in methanol as in previous protocols as we found this gave inconsistent results with 10X Genomics version 3 kits. Sorted cells are then spun down at 350rcf at 4oC for 2 min in a swinging bucket centrifuge with slow deceleration. Most of the supernatant is removed and the cells are resuspended in approximately remaining 100mL. Cells deemed to be at least 80% viable were loaded based on live cell concentration onto a Chromium X Series instrument 10x Genomics and libraries prepared using the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 10x Genomics according to manufacturer's directions. Resulting cDNA and short fragment libraries were checked for quality and quantity using a 2100 Bioanalyzer Agilent Technologies and Qubit Fluorometer Thermo Fisher Scientific. With cells captured estimated at 6 800 9 600 cells per sample  libraries were pooled and sequenced to a depth necessary to achieve at least 30 000 mean reads per cell on an Illumina NextSeq 2000 instrument utilizing RTA and instrument software versions current at the time of processing with the following paired read lengths: 28*10*10*90bp. scRNA seq 10x Genomics,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 2000,,SRP509548,,assembly:danRer11|intentional duplicate,ccndx_sibling2.bam,10X Genomics bam file,27438063300.0,304867370.0,GSM8288756 r1,0:90,A:7768441003;C:5988780076;G:6567352571;T:7111630965;N:1858685,90,,,,7768441003,5988780076,6567352571,7111630965,1858685,SRX24673329,SRS21408057,SRA1876130,"Computational Biology, Stowers Institute for Medical Research","Computational Biology, Stowers Institute for Medical Research",1,0.94768,,0.20814,,0.75452,,0.52756,,90,,B,,usable mapping rate,illumina,nextseq_v2,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2024-05-23,Larval,Larval,Lateral Line,Sensory System
32367,SRR29152188,SRX24673328,SRS21408056,SRP509548,PRJNA1115803,Stem and progenitor cell proliferation are independently regulated by cell type specific cyclinD genes,GSE268255,Transcriptome Analysis,Regeneration and homeostatic turnover of solid tissues depend on the proliferation of symmetrically dividing adult stem cells  which either remain stem cells or differentiate based on their niche position. Here we demonstrate that in zebrafish lateral line sensory organs  stem and progenitor cell proliferation are independently regulated by two cyclinD genes. Loss of ccnd2a impairs stem cell proliferation during development  while loss of ccndx disrupts hair cell progenitor proliferation but allows normal differentiation. Notably  ccnd2a can functionally replace ccndx  indicating that the respective effects of these Cyclins on proliferation are due to cell type specific expression. However  even though hair cell progenitors differentiate normally in ccndx mutants  they are mispolarized due to hes2 and Emx2 downregulation. Thus  regulated proliferation ensures that equal numbers of hair cells are polarized in opposite directions. Our study reveals cell type specific roles for cyclinD genes in regulating the different populations of symmetrically dividing cells governing organ development and regeneration  with implications for regenerative medicine and disease. Overall design: Two replicates per sibling or ccndx mutant were used,,,,ccndx sibling1,GSM8288755,,tissue:5dpf zebrafish FACS sorted lateral line cells|cell type:Homostasis|genotype:Wildtype|geo loc name:missing|collection date:missing,ccndx sibling1,Raw sequencing data were processed using 10X Genomics Cell Ranger pipeline. Reads were demultiplexed into Fastq file format using cellranger mkfastq. Genome index was built by cellranger mkref using zebrafish genome danRer11  ensembl 98 gene model. Data were aligned by STAR aligner and cell counts tables were generated using cellranger count function with default parameters. Assembly: danRer11 Supplementary files format and content: single cell  count matrix in hdf5 format  raw data in bam format.,5dpf zebrafish FACS sorted lateral line cells,All cells were collected during homeostasis  no treatments were performed.,5 dpf zebrafish were anesthetized with Tricane then split between two 70mm cell strainers Falcon and placed in 0.25% Trypsin EDTA Gibco on ice. The larvae from each strainer are then added to a 5ml round bottom tube Falcon and the tissue is broken up via slowly pipetting up and down using a pipette man and a glass pipette VWR keeping the tubes on ice. The total time from placing larvae in trypsin to finishing pipetting is approximately 8 minutes. The tissue is then filtered through 70mm filter BD Biosciences  pelleted for 5 min at 2000rpm at 4oC and then the pellet is resuspended in 1xDPBS/0.04%BSA. The pelleting and resuspension in 1xDPBS/0.04%BSA is repeated two more times. post the final pellet the cells are resuspended in 0.5mL 1xDPBS/0.04%BSA then cells are combined and put through another 70mm filter into a 5ml round bottom tube. Cells are then incubated in Draq5 1/200 dilution for 5 minutes on ice. Draq5+/mCherry+ cells were sorted by the Stowers Institute Cytometry Core Facility using a BD Influx Cell Sorter BD Biosciences  San Jose  CA. USA  with cells collected in tubes containing 10mL 1XDPBS/0.04% BSA. Cells were not fixed in methanol as in previous protocols as we found this gave inconsistent results with 10X Genomics version 3 kits. Sorted cells are then spun down at 350rcf at 4oC for 2 min in a swinging bucket centrifuge with slow deceleration. Most of the supernatant is removed and the cells are resuspended in approximately remaining 100mL. Cells deemed to be at least 80% viable were loaded based on live cell concentration onto a Chromium X Series instrument 10x Genomics and libraries prepared using the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 10x Genomics according to manufacturer’s directions. Resulting cDNA and short fragment libraries were checked for quality and quantity using a 2100 Bioanalyzer Agilent Technologies and Qubit Fluorometer Thermo Fisher Scientific. With cells captured estimated at 6 800 9 600 cells per sample  libraries were pooled and sequenced to a depth necessary to achieve at least 30 000 mean reads per cell on an Illumina NextSeq 2000 instrument utilizing RTA and instrument software versions current at the time of processing with the following paired read lengths: 28*10*10*90bp. scRNA seq 10x Genomics,5dpf zebrafish FACS sorted lateral line cells were used. Standard growing condition were used.,cell type:Homostasis|genotype:Wildtype,GSM8288755,GSM8288755: ccndx sibling1; Danio rerio; RNA Seq,GSM8288755 r1,GSM8288755,1,5 dpf zebrafish were anesthetized with Tricane then split between two 70mm cell strainers Falcon and placed in 0.25% Trypsin EDTA Gibco on ice. The larvae from each strainer are then added to a 5ml round bottom tube Falcon and the tissue is broken up via slowly pipetting up and down using a pipette man and a glass pipette VWR keeping the tubes on ice. The total time from placing larvae in trypsin to finishing pipetting is approximately 8 minutes. The tissue is then filtered through 70mm filter BD Biosciences  pelleted for 5 min at 2000rpm at 4oC and then the pellet is resuspended in 1xDPBS/0.04%BSA. The pelleting and resuspension in 1xDPBS/0.04%BSA is repeated two more times. post the final pellet the cells are resuspended in 0.5mL 1xDPBS/0.04%BSA then cells are combined and put through another 70mm filter into a 5ml round bottom tube. Cells are then incubated in Draq5 1/200 dilution for 5 minutes on ice. Draq5+/mCherry+ cells were sorted by the Stowers Institute Cytometry Core Facility using a BD Influx Cell Sorter BD Biosciences  San Jose  CA. USA  with cells collected in tubes containing 10mL 1XDPBS/0.04% BSA. Cells were not fixed in methanol as in previous protocols as we found this gave inconsistent results with 10X Genomics version 3 kits. Sorted cells are then spun down at 350rcf at 4oC for 2 min in a swinging bucket centrifuge with slow deceleration. Most of the supernatant is removed and the cells are resuspended in approximately remaining 100mL. Cells deemed to be at least 80% viable were loaded based on live cell concentration onto a Chromium X Series instrument 10x Genomics and libraries prepared using the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 10x Genomics according to manufacturer's directions. Resulting cDNA and short fragment libraries were checked for quality and quantity using a 2100 Bioanalyzer Agilent Technologies and Qubit Fluorometer Thermo Fisher Scientific. With cells captured estimated at 6 800 9 600 cells per sample  libraries were pooled and sequenced to a depth necessary to achieve at least 30 000 mean reads per cell on an Illumina NextSeq 2000 instrument utilizing RTA and instrument software versions current at the time of processing with the following paired read lengths: 28*10*10*90bp. scRNA seq 10x Genomics,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 2000,,SRP509548,,assembly:danRer11|intentional duplicate,ccndx_sibling1.bam,10X Genomics bam file,27531756630.0,305908407.0,GSM8288755 r1,0:90,A:8252775526;C:5615092533;G:6288684779;T:7373374402;N:1829390,90,,,,8252775526,5615092533,6288684779,7373374402,1829390,SRX24673328,SRS21408056,SRA1876130,"Computational Biology, Stowers Institute for Medical Research","Computational Biology, Stowers Institute for Medical Research",1,0.92077,,0.29182,,0.76524,,0.53575,,90,,B,,usable mapping rate,illumina,nextseq_v2,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2024-05-23,Larval,Larval,Lateral Line,Sensory System
32368,SRR29152189,SRX24673327,SRS21408059,SRP509548,PRJNA1115803,Stem and progenitor cell proliferation are independently regulated by cell type specific cyclinD genes,GSE268255,Transcriptome Analysis,Regeneration and homeostatic turnover of solid tissues depend on the proliferation of symmetrically dividing adult stem cells  which either remain stem cells or differentiate based on their niche position. Here we demonstrate that in zebrafish lateral line sensory organs  stem and progenitor cell proliferation are independently regulated by two cyclinD genes. Loss of ccnd2a impairs stem cell proliferation during development  while loss of ccndx disrupts hair cell progenitor proliferation but allows normal differentiation. Notably  ccnd2a can functionally replace ccndx  indicating that the respective effects of these Cyclins on proliferation are due to cell type specific expression. However  even though hair cell progenitors differentiate normally in ccndx mutants  they are mispolarized due to hes2 and Emx2 downregulation. Thus  regulated proliferation ensures that equal numbers of hair cells are polarized in opposite directions. Our study reveals cell type specific roles for cyclinD genes in regulating the different populations of symmetrically dividing cells governing organ development and regeneration  with implications for regenerative medicine and disease. Overall design: Two replicates per sibling or ccndx mutant were used,,,,ccndx mutant2,GSM8288754,,tissue:5dpf zebrafish FACS sorted lateral line cells|cell type:Homostasis|genotype:Mutant|geo loc name:missing|collection date:missing,ccndx mutant2,Raw sequencing data were processed using 10X Genomics Cell Ranger pipeline. Reads were demultiplexed into Fastq file format using cellranger mkfastq. Genome index was built by cellranger mkref using zebrafish genome danRer11  ensembl 98 gene model. Data were aligned by STAR aligner and cell counts tables were generated using cellranger count function with default parameters. Assembly: danRer11 Supplementary files format and content: single cell  count matrix in hdf5 format  raw data in bam format.,5dpf zebrafish FACS sorted lateral line cells,All cells were collected during homeostasis  no treatments were performed.,5 dpf zebrafish were anesthetized with Tricane then split between two 70mm cell strainers Falcon and placed in 0.25% Trypsin EDTA Gibco on ice. The larvae from each strainer are then added to a 5ml round bottom tube Falcon and the tissue is broken up via slowly pipetting up and down using a pipette man and a glass pipette VWR keeping the tubes on ice. The total time from placing larvae in trypsin to finishing pipetting is approximately 8 minutes. The tissue is then filtered through 70mm filter BD Biosciences  pelleted for 5 min at 2000rpm at 4oC and then the pellet is resuspended in 1xDPBS/0.04%BSA. The pelleting and resuspension in 1xDPBS/0.04%BSA is repeated two more times. post the final pellet the cells are resuspended in 0.5mL 1xDPBS/0.04%BSA then cells are combined and put through another 70mm filter into a 5ml round bottom tube. Cells are then incubated in Draq5 1/200 dilution for 5 minutes on ice. Draq5+/mCherry+ cells were sorted by the Stowers Institute Cytometry Core Facility using a BD Influx Cell Sorter BD Biosciences  San Jose  CA. USA  with cells collected in tubes containing 10mL 1XDPBS/0.04% BSA. Cells were not fixed in methanol as in previous protocols as we found this gave inconsistent results with 10X Genomics version 3 kits. Sorted cells are then spun down at 350rcf at 4oC for 2 min in a swinging bucket centrifuge with slow deceleration. Most of the supernatant is removed and the cells are resuspended in approximately remaining 100mL. Cells deemed to be at least 80% viable were loaded based on live cell concentration onto a Chromium X Series instrument 10x Genomics and libraries prepared using the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 10x Genomics according to manufacturer’s directions. Resulting cDNA and short fragment libraries were checked for quality and quantity using a 2100 Bioanalyzer Agilent Technologies and Qubit Fluorometer Thermo Fisher Scientific. With cells captured estimated at 6 800 9 600 cells per sample  libraries were pooled and sequenced to a depth necessary to achieve at least 30 000 mean reads per cell on an Illumina NextSeq 2000 instrument utilizing RTA and instrument software versions current at the time of processing with the following paired read lengths: 28*10*10*90bp. scRNA seq 10x Genomics,5dpf zebrafish FACS sorted lateral line cells were used. Standard growing condition were used.,cell type:Homostasis|genotype:Mutant,GSM8288754,GSM8288754: ccndx mutant2; Danio rerio; RNA Seq,GSM8288754 r1,GSM8288754,1,5 dpf zebrafish were anesthetized with Tricane then split between two 70mm cell strainers Falcon and placed in 0.25% Trypsin EDTA Gibco on ice. The larvae from each strainer are then added to a 5ml round bottom tube Falcon and the tissue is broken up via slowly pipetting up and down using a pipette man and a glass pipette VWR keeping the tubes on ice. The total time from placing larvae in trypsin to finishing pipetting is approximately 8 minutes. The tissue is then filtered through 70mm filter BD Biosciences  pelleted for 5 min at 2000rpm at 4oC and then the pellet is resuspended in 1xDPBS/0.04%BSA. The pelleting and resuspension in 1xDPBS/0.04%BSA is repeated two more times. post the final pellet the cells are resuspended in 0.5mL 1xDPBS/0.04%BSA then cells are combined and put through another 70mm filter into a 5ml round bottom tube. Cells are then incubated in Draq5 1/200 dilution for 5 minutes on ice. Draq5+/mCherry+ cells were sorted by the Stowers Institute Cytometry Core Facility using a BD Influx Cell Sorter BD Biosciences  San Jose  CA. USA  with cells collected in tubes containing 10mL 1XDPBS/0.04% BSA. Cells were not fixed in methanol as in previous protocols as we found this gave inconsistent results with 10X Genomics version 3 kits. Sorted cells are then spun down at 350rcf at 4oC for 2 min in a swinging bucket centrifuge with slow deceleration. Most of the supernatant is removed and the cells are resuspended in approximately remaining 100mL. Cells deemed to be at least 80% viable were loaded based on live cell concentration onto a Chromium X Series instrument 10x Genomics and libraries prepared using the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 10x Genomics according to manufacturer's directions. Resulting cDNA and short fragment libraries were checked for quality and quantity using a 2100 Bioanalyzer Agilent Technologies and Qubit Fluorometer Thermo Fisher Scientific. With cells captured estimated at 6 800 9 600 cells per sample  libraries were pooled and sequenced to a depth necessary to achieve at least 30 000 mean reads per cell on an Illumina NextSeq 2000 instrument utilizing RTA and instrument software versions current at the time of processing with the following paired read lengths: 28*10*10*90bp. scRNA seq 10x Genomics,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 2000,,SRP509548,,assembly:danRer11|intentional duplicate,ccndx_mutant2.bam,10X Genomics bam file,24590059740.0,273222886.0,GSM8288754 r1,0:90,A:6996284463;C:5349540108;G:5922688427;T:6319952971;N:1593771,90,,,,6996284463,5349540108,5922688427,6319952971,1593771,SRX24673327,SRS21408059,SRA1876130,"Computational Biology, Stowers Institute for Medical Research","Computational Biology, Stowers Institute for Medical Research",1,0.94389,,0.21693,,0.75426,,0.54926,,90,,B,,usable mapping rate,illumina,nextseq_v2,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2024-05-23,Larval,Larval,Lateral Line,Sensory System
32369,SRR29152190,SRX24673326,SRS21408058,SRP509548,PRJNA1115803,Stem and progenitor cell proliferation are independently regulated by cell type specific cyclinD genes,GSE268255,Transcriptome Analysis,Regeneration and homeostatic turnover of solid tissues depend on the proliferation of symmetrically dividing adult stem cells  which either remain stem cells or differentiate based on their niche position. Here we demonstrate that in zebrafish lateral line sensory organs  stem and progenitor cell proliferation are independently regulated by two cyclinD genes. Loss of ccnd2a impairs stem cell proliferation during development  while loss of ccndx disrupts hair cell progenitor proliferation but allows normal differentiation. Notably  ccnd2a can functionally replace ccndx  indicating that the respective effects of these Cyclins on proliferation are due to cell type specific expression. However  even though hair cell progenitors differentiate normally in ccndx mutants  they are mispolarized due to hes2 and Emx2 downregulation. Thus  regulated proliferation ensures that equal numbers of hair cells are polarized in opposite directions. Our study reveals cell type specific roles for cyclinD genes in regulating the different populations of symmetrically dividing cells governing organ development and regeneration  with implications for regenerative medicine and disease. Overall design: Two replicates per sibling or ccndx mutant were used,,,,ccndx mutant1,GSM8288753,,tissue:5dpf zebrafish FACS sorted lateral line cells|cell type:Homostasis|genotype:Mutant|geo loc name:missing|collection date:missing,ccndx mutant1,Raw sequencing data were processed using 10X Genomics Cell Ranger pipeline. Reads were demultiplexed into Fastq file format using cellranger mkfastq. Genome index was built by cellranger mkref using zebrafish genome danRer11  ensembl 98 gene model. Data were aligned by STAR aligner and cell counts tables were generated using cellranger count function with default parameters. Assembly: danRer11 Supplementary files format and content: single cell  count matrix in hdf5 format  raw data in bam format.,5dpf zebrafish FACS sorted lateral line cells,All cells were collected during homeostasis  no treatments were performed.,5 dpf zebrafish were anesthetized with Tricane then split between two 70mm cell strainers Falcon and placed in 0.25% Trypsin EDTA Gibco on ice. The larvae from each strainer are then added to a 5ml round bottom tube Falcon and the tissue is broken up via slowly pipetting up and down using a pipette man and a glass pipette VWR keeping the tubes on ice. The total time from placing larvae in trypsin to finishing pipetting is approximately 8 minutes. The tissue is then filtered through 70mm filter BD Biosciences  pelleted for 5 min at 2000rpm at 4oC and then the pellet is resuspended in 1xDPBS/0.04%BSA. The pelleting and resuspension in 1xDPBS/0.04%BSA is repeated two more times. post the final pellet the cells are resuspended in 0.5mL 1xDPBS/0.04%BSA then cells are combined and put through another 70mm filter into a 5ml round bottom tube. Cells are then incubated in Draq5 1/200 dilution for 5 minutes on ice. Draq5+/mCherry+ cells were sorted by the Stowers Institute Cytometry Core Facility using a BD Influx Cell Sorter BD Biosciences  San Jose  CA. USA  with cells collected in tubes containing 10mL 1XDPBS/0.04% BSA. Cells were not fixed in methanol as in previous protocols as we found this gave inconsistent results with 10X Genomics version 3 kits. Sorted cells are then spun down at 350rcf at 4oC for 2 min in a swinging bucket centrifuge with slow deceleration. Most of the supernatant is removed and the cells are resuspended in approximately remaining 100mL. Cells deemed to be at least 80% viable were loaded based on live cell concentration onto a Chromium X Series instrument 10x Genomics and libraries prepared using the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 10x Genomics according to manufacturer’s directions. Resulting cDNA and short fragment libraries were checked for quality and quantity using a 2100 Bioanalyzer Agilent Technologies and Qubit Fluorometer Thermo Fisher Scientific. With cells captured estimated at 6 800 9 600 cells per sample  libraries were pooled and sequenced to a depth necessary to achieve at least 30 000 mean reads per cell on an Illumina NextSeq 2000 instrument utilizing RTA and instrument software versions current at the time of processing with the following paired read lengths: 28*10*10*90bp. scRNA seq 10x Genomics,5dpf zebrafish FACS sorted lateral line cells were used. Standard growing condition were used.,cell type:Homostasis|genotype:Mutant,GSM8288753,GSM8288753: ccndx mutant1; Danio rerio; RNA Seq,GSM8288753 r1,GSM8288753,1,5 dpf zebrafish were anesthetized with Tricane then split between two 70mm cell strainers Falcon and placed in 0.25% Trypsin EDTA Gibco on ice. The larvae from each strainer are then added to a 5ml round bottom tube Falcon and the tissue is broken up via slowly pipetting up and down using a pipette man and a glass pipette VWR keeping the tubes on ice. The total time from placing larvae in trypsin to finishing pipetting is approximately 8 minutes. The tissue is then filtered through 70mm filter BD Biosciences  pelleted for 5 min at 2000rpm at 4oC and then the pellet is resuspended in 1xDPBS/0.04%BSA. The pelleting and resuspension in 1xDPBS/0.04%BSA is repeated two more times. post the final pellet the cells are resuspended in 0.5mL 1xDPBS/0.04%BSA then cells are combined and put through another 70mm filter into a 5ml round bottom tube. Cells are then incubated in Draq5 1/200 dilution for 5 minutes on ice. Draq5+/mCherry+ cells were sorted by the Stowers Institute Cytometry Core Facility using a BD Influx Cell Sorter BD Biosciences  San Jose  CA. USA  with cells collected in tubes containing 10mL 1XDPBS/0.04% BSA. Cells were not fixed in methanol as in previous protocols as we found this gave inconsistent results with 10X Genomics version 3 kits. Sorted cells are then spun down at 350rcf at 4oC for 2 min in a swinging bucket centrifuge with slow deceleration. Most of the supernatant is removed and the cells are resuspended in approximately remaining 100mL. Cells deemed to be at least 80% viable were loaded based on live cell concentration onto a Chromium X Series instrument 10x Genomics and libraries prepared using the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 10x Genomics according to manufacturer's directions. Resulting cDNA and short fragment libraries were checked for quality and quantity using a 2100 Bioanalyzer Agilent Technologies and Qubit Fluorometer Thermo Fisher Scientific. With cells captured estimated at 6 800 9 600 cells per sample  libraries were pooled and sequenced to a depth necessary to achieve at least 30 000 mean reads per cell on an Illumina NextSeq 2000 instrument utilizing RTA and instrument software versions current at the time of processing with the following paired read lengths: 28*10*10*90bp. scRNA seq 10x Genomics,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 2000,,SRP509548,,assembly:danRer11|intentional duplicate,ccndx_mutant1.bam,10X Genomics bam file,29191358880.0,324348432.0,GSM8288753 r1,0:90,A:8805522313;C:5925846684;G:6724305340;T:7733851337;N:1833206,90,,,,8805522313,5925846684,6724305340,7733851337,1833206,SRX24673326,SRS21408058,SRA1876130,"Computational Biology, Stowers Institute for Medical Research","Computational Biology, Stowers Institute for Medical Research",1,0.91635,,0.28762,,0.77488,,0.56058,,90,,B,,usable mapping rate,illumina,nextseq_v2,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2024-05-23,Larval,Larval,Lateral Line,Sensory System
34272,SRR31620797,SRX26985232,SRS23451890,SRP549631,PRJNA1194578,Tet proteins regulate differentiation potential across retinal cell types,GSE283588,Transcriptome Analysis,Tet enzymes are epigenetic modifiers that impact gene expression via 5mC to 5hmC oxidation; 5hmC generation is known to induce chromatin remodeling and promote transcriptional accessibility and therefore gene expression. Previous work demonstrated the requirement for Tet and 5hmC in early retinogenesis–revealing that Tet nulltet2 / ;tet3 /  zebrafish exhibit extensive retinal defects. Here  we leveraged scRNAseq technologies to better understand cell type specific deficits and molecular signatures underlying the tet2 / ;tet3 /  phenotype across differentiation stages. Our results revealed phenotypes in developing tet2 / ;tet3 /  retinae including delayed specification of several retinal cell types  reduced maturity across late stage tet2 / ;tet3 /  cones  expansions of immature subpopulations of both horizontal and bipolar cells  and altered biases of bipolar cell subtype fates at late differentiation stages. Together  these data support Tet proteins as regulators of cell specification  lineage fate commitment  and terminal differentiation. This study enables a better understanding of how Tet proteins impact the differentiation of many unique yet interacting cell types in a cellularly complex neuronal tissue. Overall design: Embryonic and larval eye cells were collected from 36 48 72  and 120 hpf zebrafish retinae from sibling control sibCTL and tet2 / tet3 /  retinae. Dissociated cells were analyzed via scRNAseq,,pubmed:39713311;pubmed:40140485,,72hpf dmut replicate 2,GSM8666785,,source name:eye|tissue:eye|age:72|genotype:tet2 / tet3 / |geo loc name:missing|collection date:missing,72hpf dmut replicate 2,Generation of base count matrices were generated using cellRangerversion 6.1.2. Low quality cells were removed and ambient RNA correction was performed. post ambient RNA correction  normalized logcounts were calcluated Assembly: Danio rerio.GRCz11.106 Supplementary files format and content: matrix files,eye,,Whole sibCTL and tet2 / tet3 /  eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin  with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment  in 2 separate wells  sibCTL and  tet2 / tet3 /   dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS.  Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads  and PCR amplified. post PCR amplification  cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,tissue:eye|age:72|genotype:tet2 / tet3 / ,GSM8666785,GSM8666785: 72hpf dmut replicate 2; Danio rerio; RNA Seq,GSM8666785 r1,GSM8666785,1,Whole sibCTL and tet2 / tet3 /  eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin  with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment  in 2 separate wells  sibCTL and  tet2 / tet3 /   dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS.  Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads  and PCR amplified. post PCR amplification  cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP549631,,,72-2_DMUT_Library_GRO2554A4_S12_L001_R1_001.fastq.gz 72-2_DMUT_Library_GRO2554A4_S12_L001_R2_001.fastq.gz,fastq fastq,86963831396.0,736981622.0,GSM8666785 r1,0:28 1:90,A:24668703423;C:18985727652;G:20143273422;T:23161672343;N:4454556,28,90,,,24668703423,18985727652,20143273422,23161672343,4454556,SRX26985232,SRS23451890,SRA2028272,"Gross, Ophthalmology, University of Pittsburgh","Gross, Ophthalmology, University of Pittsburgh",,,,,,,,,,,,T,B,sc-like readlen,illumina,novaseq_era,3prime,poly_a,unknown,sc,single_cell_droplet,10x,,United States,2024-12-05,Larval,Larval,Eye,Sensory System
34273,SRR31620798,SRX26985231,SRS23451889,SRP549631,PRJNA1194578,Tet proteins regulate differentiation potential across retinal cell types,GSE283588,Transcriptome Analysis,Tet enzymes are epigenetic modifiers that impact gene expression via 5mC to 5hmC oxidation; 5hmC generation is known to induce chromatin remodeling and promote transcriptional accessibility and therefore gene expression. Previous work demonstrated the requirement for Tet and 5hmC in early retinogenesis–revealing that Tet nulltet2 / ;tet3 /  zebrafish exhibit extensive retinal defects. Here  we leveraged scRNAseq technologies to better understand cell type specific deficits and molecular signatures underlying the tet2 / ;tet3 /  phenotype across differentiation stages. Our results revealed phenotypes in developing tet2 / ;tet3 /  retinae including delayed specification of several retinal cell types  reduced maturity across late stage tet2 / ;tet3 /  cones  expansions of immature subpopulations of both horizontal and bipolar cells  and altered biases of bipolar cell subtype fates at late differentiation stages. Together  these data support Tet proteins as regulators of cell specification  lineage fate commitment  and terminal differentiation. This study enables a better understanding of how Tet proteins impact the differentiation of many unique yet interacting cell types in a cellularly complex neuronal tissue. Overall design: Embryonic and larval eye cells were collected from 36 48 72  and 120 hpf zebrafish retinae from sibling control sibCTL and tet2 / tet3 /  retinae. Dissociated cells were analyzed via scRNAseq,,pubmed:39713311;pubmed:40140485,,72hpf dmut replicate 1,GSM8666784,,source name:eye|tissue:eye|age:72|genotype:tet2 / tet3 / |geo loc name:missing|collection date:missing,72hpf dmut replicate 1,Generation of base count matrices were generated using cellRangerversion 6.1.2. Low quality cells were removed and ambient RNA correction was performed. post ambient RNA correction  normalized logcounts were calcluated Assembly: Danio rerio.GRCz11.106 Supplementary files format and content: matrix files,eye,,Whole sibCTL and tet2 / tet3 /  eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin  with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment  in 2 separate wells  sibCTL and  tet2 / tet3 /   dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS.  Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads  and PCR amplified. post PCR amplification  cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,tissue:eye|age:72|genotype:tet2 / tet3 / ,GSM8666784,GSM8666784: 72hpf dmut replicate 1; Danio rerio; RNA Seq,GSM8666784 r1,GSM8666784,1,Whole sibCTL and tet2 / tet3 /  eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin  with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment  in 2 separate wells  sibCTL and  tet2 / tet3 /   dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS.  Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads  and PCR amplified. post PCR amplification  cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP549631,,,D72_1_GRO1775A20_S18_L001_R1_001.fastq.gz D72_1_GRO1775A20_S18_L001_R2_001.fastq.gz,fastq fastq,53238080296.0,451170172.0,GSM8666784 r1,0:28 1:90,A:14916063648;C:11725098152;G:12217175655;T:14377019921;N:2722920,28,90,,,14916063648,11725098152,12217175655,14377019921,2722920,SRX26985231,SRS23451889,SRA2028272,"Gross, Ophthalmology, University of Pittsburgh","Gross, Ophthalmology, University of Pittsburgh",,,,,,,,,,,,T,B,sc-like readlen,illumina,novaseq_era,3prime,poly_a,unknown,sc,single_cell_droplet,10x,,United States,2024-12-05,Larval,Larval,Eye,Sensory System
34274,SRR31620799,SRX26985230,SRS23451888,SRP549631,PRJNA1194578,Tet proteins regulate differentiation potential across retinal cell types,GSE283588,Transcriptome Analysis,Tet enzymes are epigenetic modifiers that impact gene expression via 5mC to 5hmC oxidation; 5hmC generation is known to induce chromatin remodeling and promote transcriptional accessibility and therefore gene expression. Previous work demonstrated the requirement for Tet and 5hmC in early retinogenesis–revealing that Tet nulltet2 / ;tet3 /  zebrafish exhibit extensive retinal defects. Here  we leveraged scRNAseq technologies to better understand cell type specific deficits and molecular signatures underlying the tet2 / ;tet3 /  phenotype across differentiation stages. Our results revealed phenotypes in developing tet2 / ;tet3 /  retinae including delayed specification of several retinal cell types  reduced maturity across late stage tet2 / ;tet3 /  cones  expansions of immature subpopulations of both horizontal and bipolar cells  and altered biases of bipolar cell subtype fates at late differentiation stages. Together  these data support Tet proteins as regulators of cell specification  lineage fate commitment  and terminal differentiation. This study enables a better understanding of how Tet proteins impact the differentiation of many unique yet interacting cell types in a cellularly complex neuronal tissue. Overall design: Embryonic and larval eye cells were collected from 36 48 72  and 120 hpf zebrafish retinae from sibling control sibCTL and tet2 / tet3 /  retinae. Dissociated cells were analyzed via scRNAseq,,pubmed:39713311;pubmed:40140485,,72hpf sibCTL replicate 2,GSM8666783,,source name:eye|tissue:eye|age:72|genotype:sibCTL|geo loc name:missing|collection date:missing,72hpf sibCTL replicate 2,Generation of base count matrices were generated using cellRangerversion 6.1.2. Low quality cells were removed and ambient RNA correction was performed. post ambient RNA correction  normalized logcounts were calcluated Assembly: Danio rerio.GRCz11.106 Supplementary files format and content: matrix files,eye,,Whole sibCTL and tet2 / tet3 /  eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin  with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment  in 2 separate wells  sibCTL and  tet2 / tet3 /   dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS.  Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads  and PCR amplified. post PCR amplification  cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,tissue:eye|age:72|genotype:sibCTL,GSM8666783,GSM8666783: 72hpf sibCTL replicate 2; Danio rerio; RNA Seq,GSM8666783 r1,GSM8666783,1,Whole sibCTL and tet2 / tet3 /  eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin  with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment  in 2 separate wells  sibCTL and  tet2 / tet3 /   dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS.  Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads  and PCR amplified. post PCR amplification  cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP549631,,,72-2_CTL_Library_GRO2554A3_S9_L001_R1_001.fastq.gz 72-2_CTL_Library_GRO2554A3_S9_L001_R2_001.fastq.gz,fastq fastq,92262895746.0,781888947.0,GSM8666783 r1,0:28 1:90,A:25987659581;C:20352853331;G:21566882871;T:24350747434;N:4752529,28,90,,,25987659581,20352853331,21566882871,24350747434,4752529,SRX26985230,SRS23451888,SRA2028272,"Gross, Ophthalmology, University of Pittsburgh","Gross, Ophthalmology, University of Pittsburgh",,,,,,,,,,,,T,B,sc-like readlen,illumina,novaseq_era,3prime,poly_a,unknown,sc,single_cell_droplet,10x,,United States,2024-12-05,Larval,Larval,Eye,Sensory System
34275,SRR31620800,SRX26985229,SRS23451887,SRP549631,PRJNA1194578,Tet proteins regulate differentiation potential across retinal cell types,GSE283588,Transcriptome Analysis,Tet enzymes are epigenetic modifiers that impact gene expression via 5mC to 5hmC oxidation; 5hmC generation is known to induce chromatin remodeling and promote transcriptional accessibility and therefore gene expression. Previous work demonstrated the requirement for Tet and 5hmC in early retinogenesis–revealing that Tet nulltet2 / ;tet3 /  zebrafish exhibit extensive retinal defects. Here  we leveraged scRNAseq technologies to better understand cell type specific deficits and molecular signatures underlying the tet2 / ;tet3 /  phenotype across differentiation stages. Our results revealed phenotypes in developing tet2 / ;tet3 /  retinae including delayed specification of several retinal cell types  reduced maturity across late stage tet2 / ;tet3 /  cones  expansions of immature subpopulations of both horizontal and bipolar cells  and altered biases of bipolar cell subtype fates at late differentiation stages. Together  these data support Tet proteins as regulators of cell specification  lineage fate commitment  and terminal differentiation. This study enables a better understanding of how Tet proteins impact the differentiation of many unique yet interacting cell types in a cellularly complex neuronal tissue. Overall design: Embryonic and larval eye cells were collected from 36 48 72  and 120 hpf zebrafish retinae from sibling control sibCTL and tet2 / tet3 /  retinae. Dissociated cells were analyzed via scRNAseq,,pubmed:39713311;pubmed:40140485,,72hpf sibCTL replicate 1,GSM8666782,,source name:eye|tissue:eye|age:72|genotype:sibCTL|geo loc name:missing|collection date:missing,72hpf sibCTL replicate 1,Generation of base count matrices were generated using cellRangerversion 6.1.2. Low quality cells were removed and ambient RNA correction was performed. post ambient RNA correction  normalized logcounts were calcluated Assembly: Danio rerio.GRCz11.106 Supplementary files format and content: matrix files,eye,,Whole sibCTL and tet2 / tet3 /  eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin  with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment  in 2 separate wells  sibCTL and  tet2 / tet3 /   dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS.  Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads  and PCR amplified. post PCR amplification  cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,tissue:eye|age:72|genotype:sibCTL,GSM8666782,GSM8666782: 72hpf sibCTL replicate 1; Danio rerio; RNA Seq,GSM8666782 r1,GSM8666782,1,Whole sibCTL and tet2 / tet3 /  eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin  with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment  in 2 separate wells  sibCTL and  tet2 / tet3 /   dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS.  Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads  and PCR amplified. post PCR amplification  cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP549631,,,C72_1_GRO1775A19_S16_L001_R1_001.fastq.gz C72_1_GRO1775A19_S16_L001_R2_001.fastq.gz,fastq fastq,62746229072.0,531747704.0,GSM8666782 r1,0:28 1:90,A:17508639163;C:13921967178;G:14514318917;T:16798101260;N:3202554,28,90,,,17508639163,13921967178,14514318917,16798101260,3202554,SRX26985229,SRS23451887,SRA2028272,"Gross, Ophthalmology, University of Pittsburgh","Gross, Ophthalmology, University of Pittsburgh",,,,,,,,,,,,T,B,sc-like readlen,illumina,novaseq_era,3prime,poly_a,unknown,sc,single_cell_droplet,10x,,United States,2024-12-05,Larval,Larval,Eye,Sensory System
34276,SRR31620801,SRX26985228,SRS23451886,SRP549631,PRJNA1194578,Tet proteins regulate differentiation potential across retinal cell types,GSE283588,Transcriptome Analysis,Tet enzymes are epigenetic modifiers that impact gene expression via 5mC to 5hmC oxidation; 5hmC generation is known to induce chromatin remodeling and promote transcriptional accessibility and therefore gene expression. Previous work demonstrated the requirement for Tet and 5hmC in early retinogenesis–revealing that Tet nulltet2 / ;tet3 /  zebrafish exhibit extensive retinal defects. Here  we leveraged scRNAseq technologies to better understand cell type specific deficits and molecular signatures underlying the tet2 / ;tet3 /  phenotype across differentiation stages. Our results revealed phenotypes in developing tet2 / ;tet3 /  retinae including delayed specification of several retinal cell types  reduced maturity across late stage tet2 / ;tet3 /  cones  expansions of immature subpopulations of both horizontal and bipolar cells  and altered biases of bipolar cell subtype fates at late differentiation stages. Together  these data support Tet proteins as regulators of cell specification  lineage fate commitment  and terminal differentiation. This study enables a better understanding of how Tet proteins impact the differentiation of many unique yet interacting cell types in a cellularly complex neuronal tissue. Overall design: Embryonic and larval eye cells were collected from 36 48 72  and 120 hpf zebrafish retinae from sibling control sibCTL and tet2 / tet3 /  retinae. Dissociated cells were analyzed via scRNAseq,,pubmed:39713311;pubmed:40140485,,120hpf dmut replicate 3,GSM8666781,,source name:eye|tissue:eye|age:120|genotype:tet2 / tet3 / |geo loc name:missing|collection date:missing,120hpf dmut replicate 3,Generation of base count matrices were generated using cellRangerversion 6.1.2. Low quality cells were removed and ambient RNA correction was performed. post ambient RNA correction  normalized logcounts were calcluated Assembly: Danio rerio.GRCz11.106 Supplementary files format and content: matrix files,eye,,Whole sibCTL and tet2 / tet3 /  eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin  with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment  in 2 separate wells  sibCTL and  tet2 / tet3 /   dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS.  Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads  and PCR amplified. post PCR amplification  cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,tissue:eye|age:120|genotype:tet2 / tet3 / ,GSM8666781,GSM8666781: 120hpf dmut replicate 3; Danio rerio; RNA Seq,GSM8666781 r1,GSM8666781,1,Whole sibCTL and tet2 / tet3 /  eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin  with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment  in 2 separate wells  sibCTL and  tet2 / tet3 /   dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS.  Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads  and PCR amplified. post PCR amplification  cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP549631,,,120-3_DMUT_Library_GRO2554A6_S17_L001_R1_001.fastq.gz 120-3_DMUT_Library_GRO2554A6_S17_L001_R2_001.fastq.gz,fastq fastq,78060738122.0,661531679.0,GSM8666781 r1,0:28 1:90,A:22149025023;C:16969925464;G:17983050030;T:20954744171;N:3993434,28,90,,,22149025023,16969925464,17983050030,20954744171,3993434,SRX26985228,SRS23451886,SRA2028272,"Gross, Ophthalmology, University of Pittsburgh","Gross, Ophthalmology, University of Pittsburgh",,,,,,,,,,,,T,B,sc-like readlen,illumina,novaseq_era,3prime,poly_a,unknown,sc,single_cell_droplet,10x,,United States,2024-12-05,Larval,Larval,Eye,Sensory System
34277,SRR31620802,SRX26985227,SRS23451885,SRP549631,PRJNA1194578,Tet proteins regulate differentiation potential across retinal cell types,GSE283588,Transcriptome Analysis,Tet enzymes are epigenetic modifiers that impact gene expression via 5mC to 5hmC oxidation; 5hmC generation is known to induce chromatin remodeling and promote transcriptional accessibility and therefore gene expression. Previous work demonstrated the requirement for Tet and 5hmC in early retinogenesis–revealing that Tet nulltet2 / ;tet3 /  zebrafish exhibit extensive retinal defects. Here  we leveraged scRNAseq technologies to better understand cell type specific deficits and molecular signatures underlying the tet2 / ;tet3 /  phenotype across differentiation stages. Our results revealed phenotypes in developing tet2 / ;tet3 /  retinae including delayed specification of several retinal cell types  reduced maturity across late stage tet2 / ;tet3 /  cones  expansions of immature subpopulations of both horizontal and bipolar cells  and altered biases of bipolar cell subtype fates at late differentiation stages. Together  these data support Tet proteins as regulators of cell specification  lineage fate commitment  and terminal differentiation. This study enables a better understanding of how Tet proteins impact the differentiation of many unique yet interacting cell types in a cellularly complex neuronal tissue. Overall design: Embryonic and larval eye cells were collected from 36 48 72  and 120 hpf zebrafish retinae from sibling control sibCTL and tet2 / tet3 /  retinae. Dissociated cells were analyzed via scRNAseq,,pubmed:39713311;pubmed:40140485,,120hpf dmut replicate 2,GSM8666780,,source name:eye|tissue:eye|age:120|genotype:tet2 / tet3 / |geo loc name:missing|collection date:missing,120hpf dmut replicate 2,Generation of base count matrices were generated using cellRangerversion 6.1.2. Low quality cells were removed and ambient RNA correction was performed. post ambient RNA correction  normalized logcounts were calcluated Assembly: Danio rerio.GRCz11.106 Supplementary files format and content: matrix files,eye,,Whole sibCTL and tet2 / tet3 /  eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin  with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment  in 2 separate wells  sibCTL and  tet2 / tet3 /   dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS.  Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads  and PCR amplified. post PCR amplification  cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,tissue:eye|age:120|genotype:tet2 / tet3 / ,GSM8666780,GSM8666780: 120hpf dmut replicate 2; Danio rerio; RNA Seq,GSM8666780 r1,GSM8666780,1,Whole sibCTL and tet2 / tet3 /  eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin  with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment  in 2 separate wells  sibCTL and  tet2 / tet3 /   dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS.  Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads  and PCR amplified. post PCR amplification  cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP549631,,,120_2DMUT_LIBRARY_GRO2455A8_S14_L001_R1_001.fastq.gz 120_2DMUT_LIBRARY_GRO2455A8_S14_L001_R2_001.fastq.gz,fastq fastq,75309468058.0,638215831.0,GSM8666780 r1,0:28 1:90,A:21252598025;C:16527675199;G:17525784489;T:19999551980;N:3858365,28,90,,,21252598025,16527675199,17525784489,19999551980,3858365,SRX26985227,SRS23451885,SRA2028272,"Gross, Ophthalmology, University of Pittsburgh","Gross, Ophthalmology, University of Pittsburgh",,,,,,,,,,,,T,B,sc-like readlen,illumina,novaseq_era,3prime,poly_a,unknown,sc,single_cell_droplet,10x,,United States,2024-12-05,Larval,Larval,Eye,Sensory System
34278,SRR31620803,SRX26985226,SRS23451884,SRP549631,PRJNA1194578,Tet proteins regulate differentiation potential across retinal cell types,GSE283588,Transcriptome Analysis,Tet enzymes are epigenetic modifiers that impact gene expression via 5mC to 5hmC oxidation; 5hmC generation is known to induce chromatin remodeling and promote transcriptional accessibility and therefore gene expression. Previous work demonstrated the requirement for Tet and 5hmC in early retinogenesis–revealing that Tet nulltet2 / ;tet3 /  zebrafish exhibit extensive retinal defects. Here  we leveraged scRNAseq technologies to better understand cell type specific deficits and molecular signatures underlying the tet2 / ;tet3 /  phenotype across differentiation stages. Our results revealed phenotypes in developing tet2 / ;tet3 /  retinae including delayed specification of several retinal cell types  reduced maturity across late stage tet2 / ;tet3 /  cones  expansions of immature subpopulations of both horizontal and bipolar cells  and altered biases of bipolar cell subtype fates at late differentiation stages. Together  these data support Tet proteins as regulators of cell specification  lineage fate commitment  and terminal differentiation. This study enables a better understanding of how Tet proteins impact the differentiation of many unique yet interacting cell types in a cellularly complex neuronal tissue. Overall design: Embryonic and larval eye cells were collected from 36 48 72  and 120 hpf zebrafish retinae from sibling control sibCTL and tet2 / tet3 /  retinae. Dissociated cells were analyzed via scRNAseq,,pubmed:39713311;pubmed:40140485,,120hpf dmut replicate 1,GSM8666779,,source name:eye|tissue:eye|age:120|genotype:tet2 / tet3 / |geo loc name:missing|collection date:missing,120hpf dmut replicate 1,Generation of base count matrices were generated using cellRangerversion 6.1.2. Low quality cells were removed and ambient RNA correction was performed. post ambient RNA correction  normalized logcounts were calcluated Assembly: Danio rerio.GRCz11.106 Supplementary files format and content: matrix files,eye,,Whole sibCTL and tet2 / tet3 /  eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin  with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment  in 2 separate wells  sibCTL and  tet2 / tet3 /   dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS.  Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads  and PCR amplified. post PCR amplification  cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,tissue:eye|age:120|genotype:tet2 / tet3 / ,GSM8666779,GSM8666779: 120hpf dmut replicate 1; Danio rerio; RNA Seq,GSM8666779 r1,GSM8666779,1,Whole sibCTL and tet2 / tet3 /  eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin  with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment  in 2 separate wells  sibCTL and  tet2 / tet3 /   dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS.  Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads  and PCR amplified. post PCR amplification  cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP549631,,,120_1_DMUT_library_GRO2404A4_S7_L001_R1_001.fastq.gz 120_1_DMUT_library_GRO2404A4_S7_L001_R2_001.fastq.gz,fastq fastq,43416581576.0,367937132.0,GSM8666779 r1,0:28 1:90,A:12280582960;C:9514351863;G:10067617414;T:11551801481;N:2227858,28,90,,,12280582960,9514351863,10067617414,11551801481,2227858,SRX26985226,SRS23451884,SRA2028272,"Gross, Ophthalmology, University of Pittsburgh","Gross, Ophthalmology, University of Pittsburgh",,,,,,,,,,,,T,B,sc-like readlen,illumina,novaseq_era,3prime,poly_a,unknown,sc,single_cell_droplet,10x,,United States,2024-12-05,Larval,Larval,Eye,Sensory System
34279,SRR31620804,SRX26985225,SRS23451883,SRP549631,PRJNA1194578,Tet proteins regulate differentiation potential across retinal cell types,GSE283588,Transcriptome Analysis,Tet enzymes are epigenetic modifiers that impact gene expression via 5mC to 5hmC oxidation; 5hmC generation is known to induce chromatin remodeling and promote transcriptional accessibility and therefore gene expression. Previous work demonstrated the requirement for Tet and 5hmC in early retinogenesis–revealing that Tet nulltet2 / ;tet3 /  zebrafish exhibit extensive retinal defects. Here  we leveraged scRNAseq technologies to better understand cell type specific deficits and molecular signatures underlying the tet2 / ;tet3 /  phenotype across differentiation stages. Our results revealed phenotypes in developing tet2 / ;tet3 /  retinae including delayed specification of several retinal cell types  reduced maturity across late stage tet2 / ;tet3 /  cones  expansions of immature subpopulations of both horizontal and bipolar cells  and altered biases of bipolar cell subtype fates at late differentiation stages. Together  these data support Tet proteins as regulators of cell specification  lineage fate commitment  and terminal differentiation. This study enables a better understanding of how Tet proteins impact the differentiation of many unique yet interacting cell types in a cellularly complex neuronal tissue. Overall design: Embryonic and larval eye cells were collected from 36 48 72  and 120 hpf zebrafish retinae from sibling control sibCTL and tet2 / tet3 /  retinae. Dissociated cells were analyzed via scRNAseq,,pubmed:39713311;pubmed:40140485,,120hpf sibCTL replicate 3,GSM8666778,,source name:eye|tissue:eye|age:120|genotype:sibCTL|geo loc name:missing|collection date:missing,120hpf sibCTL replicate 3,Generation of base count matrices were generated using cellRangerversion 6.1.2. Low quality cells were removed and ambient RNA correction was performed. post ambient RNA correction  normalized logcounts were calcluated Assembly: Danio rerio.GRCz11.106 Supplementary files format and content: matrix files,eye,,Whole sibCTL and tet2 / tet3 /  eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin  with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment  in 2 separate wells  sibCTL and  tet2 / tet3 /   dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS.  Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads  and PCR amplified. post PCR amplification  cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,tissue:eye|age:120|genotype:sibCTL,GSM8666778,GSM8666778: 120hpf sibCTL replicate 3; Danio rerio; RNA Seq,GSM8666778 r1,GSM8666778,1,Whole sibCTL and tet2 / tet3 /  eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin  with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment  in 2 separate wells  sibCTL and  tet2 / tet3 /   dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS.  Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads  and PCR amplified. post PCR amplification  cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP549631,,,120-3_CTL_Library_GRO2554A5_S15_L001_R1_001.fastq.gz 120-3_CTL_Library_GRO2554A5_S15_L001_R2_001.fastq.gz,fastq fastq,72305032770.0,612754515.0,GSM8666778 r1,0:28 1:90,A:20458142581;C:15911862318;G:17077955694;T:18853377726;N:3694451,28,90,,,20458142581,15911862318,17077955694,18853377726,3694451,SRX26985225,SRS23451883,SRA2028272,"Gross, Ophthalmology, University of Pittsburgh","Gross, Ophthalmology, University of Pittsburgh",,,,,,,,,,,,T,B,sc-like readlen,illumina,novaseq_era,3prime,poly_a,unknown,sc,single_cell_droplet,10x,,United States,2024-12-05,Larval,Larval,Eye,Sensory System
34280,SRR31620805,SRX26985224,SRS23451882,SRP549631,PRJNA1194578,Tet proteins regulate differentiation potential across retinal cell types,GSE283588,Transcriptome Analysis,Tet enzymes are epigenetic modifiers that impact gene expression via 5mC to 5hmC oxidation; 5hmC generation is known to induce chromatin remodeling and promote transcriptional accessibility and therefore gene expression. Previous work demonstrated the requirement for Tet and 5hmC in early retinogenesis–revealing that Tet nulltet2 / ;tet3 /  zebrafish exhibit extensive retinal defects. Here  we leveraged scRNAseq technologies to better understand cell type specific deficits and molecular signatures underlying the tet2 / ;tet3 /  phenotype across differentiation stages. Our results revealed phenotypes in developing tet2 / ;tet3 /  retinae including delayed specification of several retinal cell types  reduced maturity across late stage tet2 / ;tet3 /  cones  expansions of immature subpopulations of both horizontal and bipolar cells  and altered biases of bipolar cell subtype fates at late differentiation stages. Together  these data support Tet proteins as regulators of cell specification  lineage fate commitment  and terminal differentiation. This study enables a better understanding of how Tet proteins impact the differentiation of many unique yet interacting cell types in a cellularly complex neuronal tissue. Overall design: Embryonic and larval eye cells were collected from 36 48 72  and 120 hpf zebrafish retinae from sibling control sibCTL and tet2 / tet3 /  retinae. Dissociated cells were analyzed via scRNAseq,,pubmed:39713311;pubmed:40140485,,120hpf sibCTL replicate 2,GSM8666777,,source name:eye|tissue:eye|age:120|genotype:sibCTL|geo loc name:missing|collection date:missing,120hpf sibCTL replicate 2,Generation of base count matrices were generated using cellRangerversion 6.1.2. Low quality cells were removed and ambient RNA correction was performed. post ambient RNA correction  normalized logcounts were calcluated Assembly: Danio rerio.GRCz11.106 Supplementary files format and content: matrix files,eye,,Whole sibCTL and tet2 / tet3 /  eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin  with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment  in 2 separate wells  sibCTL and  tet2 / tet3 /   dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS.  Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads  and PCR amplified. post PCR amplification  cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,tissue:eye|age:120|genotype:sibCTL,GSM8666777,GSM8666777: 120hpf sibCTL replicate 2; Danio rerio; RNA Seq,GSM8666777 r1,GSM8666777,1,Whole sibCTL and tet2 / tet3 /  eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin  with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment  in 2 separate wells  sibCTL and  tet2 / tet3 /   dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS.  Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads  and PCR amplified. post PCR amplification  cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP549631,,,120_2CTL_LIBRARY_GRO2455A7_S11_L001_R1_001.fastq.gz 120_2CTL_LIBRARY_GRO2455A7_S11_L001_R2_001.fastq.gz,fastq fastq,73544797056.0,623260992.0,GSM8666777 r1,0:28 1:90,A:20984077687;C:16373905014;G:18186271681;T:17996787181;N:3755493,28,90,,,20984077687,16373905014,18186271681,17996787181,3755493,SRX26985224,SRS23451882,SRA2028272,"Gross, Ophthalmology, University of Pittsburgh","Gross, Ophthalmology, University of Pittsburgh",,,,,,,,,,,,T,B,sc-like readlen,illumina,novaseq_era,3prime,poly_a,unknown,sc,single_cell_droplet,10x,,United States,2024-12-05,Larval,Larval,Eye,Sensory System
34281,SRR31620806,SRX26985223,SRS23451881,SRP549631,PRJNA1194578,Tet proteins regulate differentiation potential across retinal cell types,GSE283588,Transcriptome Analysis,Tet enzymes are epigenetic modifiers that impact gene expression via 5mC to 5hmC oxidation; 5hmC generation is known to induce chromatin remodeling and promote transcriptional accessibility and therefore gene expression. Previous work demonstrated the requirement for Tet and 5hmC in early retinogenesis–revealing that Tet nulltet2 / ;tet3 /  zebrafish exhibit extensive retinal defects. Here  we leveraged scRNAseq technologies to better understand cell type specific deficits and molecular signatures underlying the tet2 / ;tet3 /  phenotype across differentiation stages. Our results revealed phenotypes in developing tet2 / ;tet3 /  retinae including delayed specification of several retinal cell types  reduced maturity across late stage tet2 / ;tet3 /  cones  expansions of immature subpopulations of both horizontal and bipolar cells  and altered biases of bipolar cell subtype fates at late differentiation stages. Together  these data support Tet proteins as regulators of cell specification  lineage fate commitment  and terminal differentiation. This study enables a better understanding of how Tet proteins impact the differentiation of many unique yet interacting cell types in a cellularly complex neuronal tissue. Overall design: Embryonic and larval eye cells were collected from 36 48 72  and 120 hpf zebrafish retinae from sibling control sibCTL and tet2 / tet3 /  retinae. Dissociated cells were analyzed via scRNAseq,,pubmed:39713311;pubmed:40140485,,120hpf sibCTL replicate 1,GSM8666776,,source name:eye|tissue:eye|age:120|genotype:sibCTL|geo loc name:missing|collection date:missing,120hpf sibCTL replicate 1,Generation of base count matrices were generated using cellRangerversion 6.1.2. Low quality cells were removed and ambient RNA correction was performed. post ambient RNA correction  normalized logcounts were calcluated Assembly: Danio rerio.GRCz11.106 Supplementary files format and content: matrix files,eye,,Whole sibCTL and tet2 / tet3 /  eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin  with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment  in 2 separate wells  sibCTL and  tet2 / tet3 /   dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS.  Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads  and PCR amplified. post PCR amplification  cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,tissue:eye|age:120|genotype:sibCTL,GSM8666776,GSM8666776: 120hpf sibCTL replicate 1; Danio rerio; RNA Seq,GSM8666776 r1,GSM8666776,1,Whole sibCTL and tet2 / tet3 /  eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin  with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment  in 2 separate wells  sibCTL and  tet2 / tet3 /   dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS.  Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads  and PCR amplified. post PCR amplification  cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP549631,,,120_1_CTL_library_GRO2404A3_S3_L001_R1_001.fastq.gz 120_1_CTL_library_GRO2404A3_S3_L001_R2_001.fastq.gz,fastq fastq,41316273380.0,350137910.0,GSM8666776 r1,0:28 1:90,A:11480318362;C:9330748906;G:9904812333;T:10598496608;N:1897171,28,90,,,11480318362,9330748906,9904812333,10598496608,1897171,SRX26985223,SRS23451881,SRA2028272,"Gross, Ophthalmology, University of Pittsburgh","Gross, Ophthalmology, University of Pittsburgh",,,,,,,,,,,,T,B,sc-like readlen,illumina,novaseq_era,3prime,poly_a,unknown,sc,single_cell_droplet,10x,,United States,2024-12-05,Larval,Larval,Eye,Sensory System
34913,SRR32335084,SRX27670022,SRS24074877,SRP563805,PRJNA1223441,A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression ,GSE289562,Transcriptome Analysis,We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf.  This work will be published as Le et al.  2025: A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression. Heterozygous deletions predicting haploinsufficiency for the Cysteine Rich Motor Neuron 1 CRIM1 gene have been identified in two families with macrophthalmia  colobomatous  with microcornea MACOM  an autosomal dominant trait. Crim1 encodes a type I transmembrane protein that is expressed at the cell membrane of lens epithelial and fiber cells at the stage of lens pit formation. Decreased Crim1 expression in the mouse reduced the number of lens epithelial cells and caused defective adhesion between lens epithelial cells and between the epithelial and fiber cells. We present three patients with heterozygous deletions and truncating variants predicted to result in haploinsufficiency for CRIM1 as further evidence for the role of this gene in eye defects  including retinal coloboma  optic pallor  and glaucoma. We used Clustered Regularly Interspaced Short Palindromic Repeats CRISPR/Cas9 to make a stable Danio rerio model of crim1 deficiency  generating zebrafish that were homozygous for a 2 basepair deletion  c.339 340delCT p.Leu112Leufs*3  in crim1. Homozygous  crim1 /  larvae demonstrated smaller eyes and small and misshapen lenses compared to controls  but we did not observe colobomas. Bulk RNA Seq using dissected eyes from crim1 /  larvae and controls at 72 hpf showed significant downregulation of crim1 and chloride intracellular channel 4 clic4 and upregulation of fibroblast growth factor 1b fgf1b and complement component 1  q subcomponent c1q  amongst other dysregulated genes. Our work strengthens the association between haploinsufficiency for CRIM1 and eye defects and characterizes a stable model of crim1 loss of function for future research. Overall design: We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf. Wildtype and crim1 /  have two replicates.,,pubmed:40114969,,crim1 knockdown whole eyes  72 hpf  sample 3,GSM8794307,,source name:Whole eye|tissue:Whole eye|cell type:All|genotype:crim1 / |geo loc name:missing|collection date:missing,crim1 knockdown whole eyes  72 hpf  sample 3,The quality of raw single end reads of RNA Seq dataset was tested by FastQC http:// www.bioinformatics.babraham.ac.uk/projects/fastqc/ to evaluate the per base sequence quality  quality scores  sequence length distribution and overrepresented adapter/primer sequences.  post quality control with FastQC  data trimming and clipping was performed using BBDuk and aligned against the zebrafish reference genome GRCz11 using STAR. Alignment results were then assessed to check the quality with Qualimap. RSEM was used to quantify genes. Finally  DESeq2 was used to do the downstream analysis for differentially expressed genes. Statistically significant differentially expressed genes DEGs with log2 fold change greater than ±0.5 with an adjusted p value ≤0.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM ≥1 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script. Principal component analysis PCA was performed Assembly: GRCz11 Supplementary files format and content: Excel; Statistically significant differentially expressed genes DEGs with log2 fold change greater than ±0.5 with an adjusted p value ≤0.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM ≥1 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script.,Whole eye,,Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed ‘bulk’ RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation® RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5′ end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3′ end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.,,tissue:Whole eye|cell type:All|genotype:crim1 / ,GSM8794307,GSM8794307: crim1 knockdown whole eyes  72 hpf  sample 3; Danio rerio; RNA Seq,GSM8794307 r1,GSM8794307,1,Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed 'bulk' RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation® RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5′ end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3′ end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP563805,,,crim1-dpf-3_S15_L002_R1_001.fastq.gz,fastq,3331893906.0,50483241.0,GSM8794307 r1,0:66,A:873905674;C:752237090;G:742087342;T:963442356;N:221444,66,,,,873905674,752237090,742087342,963442356,221444,SRX27670022,SRS24074877,,,Cincinnati Children's Hospital Medical Center,,,,,,,,,,,,B,,usable mapping rate,illumina,hiseq_era,unknown,random_priming,unknown,bulk,bulk,bulk,,United States,2025-02-13,Larval,Larval,Eye,Sensory System
34914,SRR32335085,SRX27670021,SRS24074876,SRP563805,PRJNA1223441,A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression ,GSE289562,Transcriptome Analysis,We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf.  This work will be published as Le et al.  2025: A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression. Heterozygous deletions predicting haploinsufficiency for the Cysteine Rich Motor Neuron 1 CRIM1 gene have been identified in two families with macrophthalmia  colobomatous  with microcornea MACOM  an autosomal dominant trait. Crim1 encodes a type I transmembrane protein that is expressed at the cell membrane of lens epithelial and fiber cells at the stage of lens pit formation. Decreased Crim1 expression in the mouse reduced the number of lens epithelial cells and caused defective adhesion between lens epithelial cells and between the epithelial and fiber cells. We present three patients with heterozygous deletions and truncating variants predicted to result in haploinsufficiency for CRIM1 as further evidence for the role of this gene in eye defects  including retinal coloboma  optic pallor  and glaucoma. We used Clustered Regularly Interspaced Short Palindromic Repeats CRISPR/Cas9 to make a stable Danio rerio model of crim1 deficiency  generating zebrafish that were homozygous for a 2 basepair deletion  c.339 340delCT p.Leu112Leufs*3  in crim1. Homozygous  crim1 /  larvae demonstrated smaller eyes and small and misshapen lenses compared to controls  but we did not observe colobomas. Bulk RNA Seq using dissected eyes from crim1 /  larvae and controls at 72 hpf showed significant downregulation of crim1 and chloride intracellular channel 4 clic4 and upregulation of fibroblast growth factor 1b fgf1b and complement component 1  q subcomponent c1q  amongst other dysregulated genes. Our work strengthens the association between haploinsufficiency for CRIM1 and eye defects and characterizes a stable model of crim1 loss of function for future research. Overall design: We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf. Wildtype and crim1 /  have two replicates.,,pubmed:40114969,,crim1 knockdown whole eyes  72 hpf  sample 2,GSM8794306,,source name:Whole eye|tissue:Whole eye|cell type:All|genotype:crim1 / |geo loc name:missing|collection date:missing,crim1 knockdown whole eyes  72 hpf  sample 2,The quality of raw single end reads of RNA Seq dataset was tested by FastQC http:// www.bioinformatics.babraham.ac.uk/projects/fastqc/ to evaluate the per base sequence quality  quality scores  sequence length distribution and overrepresented adapter/primer sequences.  post quality control with FastQC  data trimming and clipping was performed using BBDuk and aligned against the zebrafish reference genome GRCz11 using STAR. Alignment results were then assessed to check the quality with Qualimap. RSEM was used to quantify genes. Finally  DESeq2 was used to do the downstream analysis for differentially expressed genes. Statistically significant differentially expressed genes DEGs with log2 fold change greater than ±0.5 with an adjusted p value ≤0.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM ≥1 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script. Principal component analysis PCA was performed Assembly: GRCz11 Supplementary files format and content: Excel; Statistically significant differentially expressed genes DEGs with log2 fold change greater than ±0.5 with an adjusted p value ≤0.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM ≥1 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script.,Whole eye,,Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed ‘bulk’ RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation® RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5′ end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3′ end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.,,tissue:Whole eye|cell type:All|genotype:crim1 / ,GSM8794306,GSM8794306: crim1 knockdown whole eyes  72 hpf  sample 2; Danio rerio; RNA Seq,GSM8794306 r1,GSM8794306,1,Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed 'bulk' RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation® RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5′ end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3′ end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP563805,,,crim1-dpf-2_S11_L002_R1_001.fastq.gz,fastq,3491059044.0,52894834.0,GSM8794306 r1,0:66,A:907771486;C:794245897;G:783248243;T:1005559302;N:234116,66,,,,907771486,794245897,783248243,1005559302,234116,SRX27670021,SRS24074876,,,Cincinnati Children's Hospital Medical Center,,,,,,,,,,,,B,,usable mapping rate,illumina,hiseq_era,unknown,random_priming,unknown,bulk,bulk,bulk,,United States,2025-02-13,Larval,Larval,Eye,Sensory System
34915,SRR32335086,SRX27670020,SRS24074875,SRP563805,PRJNA1223441,A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression ,GSE289562,Transcriptome Analysis,We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf.  This work will be published as Le et al.  2025: A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression. Heterozygous deletions predicting haploinsufficiency for the Cysteine Rich Motor Neuron 1 CRIM1 gene have been identified in two families with macrophthalmia  colobomatous  with microcornea MACOM  an autosomal dominant trait. Crim1 encodes a type I transmembrane protein that is expressed at the cell membrane of lens epithelial and fiber cells at the stage of lens pit formation. Decreased Crim1 expression in the mouse reduced the number of lens epithelial cells and caused defective adhesion between lens epithelial cells and between the epithelial and fiber cells. We present three patients with heterozygous deletions and truncating variants predicted to result in haploinsufficiency for CRIM1 as further evidence for the role of this gene in eye defects  including retinal coloboma  optic pallor  and glaucoma. We used Clustered Regularly Interspaced Short Palindromic Repeats CRISPR/Cas9 to make a stable Danio rerio model of crim1 deficiency  generating zebrafish that were homozygous for a 2 basepair deletion  c.339 340delCT p.Leu112Leufs*3  in crim1. Homozygous  crim1 /  larvae demonstrated smaller eyes and small and misshapen lenses compared to controls  but we did not observe colobomas. Bulk RNA Seq using dissected eyes from crim1 /  larvae and controls at 72 hpf showed significant downregulation of crim1 and chloride intracellular channel 4 clic4 and upregulation of fibroblast growth factor 1b fgf1b and complement component 1  q subcomponent c1q  amongst other dysregulated genes. Our work strengthens the association between haploinsufficiency for CRIM1 and eye defects and characterizes a stable model of crim1 loss of function for future research. Overall design: We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf. Wildtype and crim1 /  have two replicates.,,pubmed:40114969,,crim1 knockdown whole eyes  72 hpf  sample 1,GSM8794305,,source name:Whole eye|tissue:Whole eye|cell type:All|genotype:crim1 /. |geo loc name:missing|collection date:missing,crim1 knockdown whole eyes  72 hpf  sample 1,The quality of raw single end reads of RNA Seq dataset was tested by FastQC http:// www.bioinformatics.babraham.ac.uk/projects/fastqc/ to evaluate the per base sequence quality  quality scores  sequence length distribution and overrepresented adapter/primer sequences.  post quality control with FastQC  data trimming and clipping was performed using BBDuk and aligned against the zebrafish reference genome GRCz11 using STAR. Alignment results were then assessed to check the quality with Qualimap. RSEM was used to quantify genes. Finally  DESeq2 was used to do the downstream analysis for differentially expressed genes. Statistically significant differentially expressed genes DEGs with log2 fold change greater than ±0.5 with an adjusted p value ≤0.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM ≥1 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script. Principal component analysis PCA was performed Assembly: GRCz11 Supplementary files format and content: Excel; Statistically significant differentially expressed genes DEGs with log2 fold change greater than ±0.5 with an adjusted p value ≤0.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM ≥1 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script.,Whole eye,,Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed ‘bulk’ RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation® RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5′ end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3′ end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.,,tissue:Whole eye|cell type:All|genotype:crim1 /. ,GSM8794305,GSM8794305: crim1 knockdown whole eyes  72 hpf  sample 1; Danio rerio; RNA Seq,GSM8794305 r1,GSM8794305,1,Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed 'bulk' RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation® RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5′ end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3′ end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP563805,,,crim1-dpf-1_S16_L003_R1_001.fastq.gz,fastq,2751667380.0,41691930.0,GSM8794305 r1,0:66,A:714065903;C:618520760;G:607652792;T:811160146;N:267779,66,,,,714065903,618520760,607652792,811160146,267779,SRX27670020,SRS24074875,,,Cincinnati Children's Hospital Medical Center,,,,,,,,,,,,B,,usable mapping rate,illumina,hiseq_era,unknown,random_priming,unknown,bulk,bulk,bulk,,United States,2025-02-13,Larval,Larval,Eye,Sensory System
34916,SRR32335087,SRX27670019,SRS24074874,SRP563805,PRJNA1223441,A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression ,GSE289562,Transcriptome Analysis,We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf.  This work will be published as Le et al.  2025: A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression. Heterozygous deletions predicting haploinsufficiency for the Cysteine Rich Motor Neuron 1 CRIM1 gene have been identified in two families with macrophthalmia  colobomatous  with microcornea MACOM  an autosomal dominant trait. Crim1 encodes a type I transmembrane protein that is expressed at the cell membrane of lens epithelial and fiber cells at the stage of lens pit formation. Decreased Crim1 expression in the mouse reduced the number of lens epithelial cells and caused defective adhesion between lens epithelial cells and between the epithelial and fiber cells. We present three patients with heterozygous deletions and truncating variants predicted to result in haploinsufficiency for CRIM1 as further evidence for the role of this gene in eye defects  including retinal coloboma  optic pallor  and glaucoma. We used Clustered Regularly Interspaced Short Palindromic Repeats CRISPR/Cas9 to make a stable Danio rerio model of crim1 deficiency  generating zebrafish that were homozygous for a 2 basepair deletion  c.339 340delCT p.Leu112Leufs*3  in crim1. Homozygous  crim1 /  larvae demonstrated smaller eyes and small and misshapen lenses compared to controls  but we did not observe colobomas. Bulk RNA Seq using dissected eyes from crim1 /  larvae and controls at 72 hpf showed significant downregulation of crim1 and chloride intracellular channel 4 clic4 and upregulation of fibroblast growth factor 1b fgf1b and complement component 1  q subcomponent c1q  amongst other dysregulated genes. Our work strengthens the association between haploinsufficiency for CRIM1 and eye defects and characterizes a stable model of crim1 loss of function for future research. Overall design: We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf. Wildtype and crim1 /  have two replicates.,,pubmed:40114969,,wildtype whole eyes  72 hpf  sample 3,GSM8794304,,source name:Whole eye|tissue:Whole eye|cell type:All|genotype:Wildtype|geo loc name:missing|collection date:missing,wildtype whole eyes  72 hpf  sample 3,The quality of raw single end reads of RNA Seq dataset was tested by FastQC http:// www.bioinformatics.babraham.ac.uk/projects/fastqc/ to evaluate the per base sequence quality  quality scores  sequence length distribution and overrepresented adapter/primer sequences.  post quality control with FastQC  data trimming and clipping was performed using BBDuk and aligned against the zebrafish reference genome GRCz11 using STAR. Alignment results were then assessed to check the quality with Qualimap. RSEM was used to quantify genes. Finally  DESeq2 was used to do the downstream analysis for differentially expressed genes. Statistically significant differentially expressed genes DEGs with log2 fold change greater than ±0.5 with an adjusted p value ≤0.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM ≥1 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script. Principal component analysis PCA was performed Assembly: GRCz11 Supplementary files format and content: Excel; Statistically significant differentially expressed genes DEGs with log2 fold change greater than ±0.5 with an adjusted p value ≤0.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM ≥1 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script.,Whole eye,,Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed ‘bulk’ RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation® RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5′ end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3′ end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.,,tissue:Whole eye|cell type:All|genotype:Wildtype,GSM8794304,GSM8794304: wildtype whole eyes  72 hpf  sample 3; Danio rerio; RNA Seq,GSM8794304 r1,GSM8794304,1,Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed 'bulk' RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation® RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5′ end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3′ end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP563805,,,crim1-ctr-3_S12_L002_R1_001.fastq.gz,fastq,2881865976.0,43664636.0,GSM8794304 r1,0:66,A:759373846;C:644323432;G:637932338;T:840046555;N:189805,66,,,,759373846,644323432,637932338,840046555,189805,SRX27670019,SRS24074874,,,Cincinnati Children's Hospital Medical Center,,,,,,,,,,,,B,,usable mapping rate,illumina,hiseq_era,unknown,random_priming,unknown,bulk,bulk,bulk,,United States,2025-02-13,Larval,Larval,Eye,Sensory System
34917,SRR32335088,SRX27670018,SRS24074873,SRP563805,PRJNA1223441,A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression ,GSE289562,Transcriptome Analysis,We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf.  This work will be published as Le et al.  2025: A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression. Heterozygous deletions predicting haploinsufficiency for the Cysteine Rich Motor Neuron 1 CRIM1 gene have been identified in two families with macrophthalmia  colobomatous  with microcornea MACOM  an autosomal dominant trait. Crim1 encodes a type I transmembrane protein that is expressed at the cell membrane of lens epithelial and fiber cells at the stage of lens pit formation. Decreased Crim1 expression in the mouse reduced the number of lens epithelial cells and caused defective adhesion between lens epithelial cells and between the epithelial and fiber cells. We present three patients with heterozygous deletions and truncating variants predicted to result in haploinsufficiency for CRIM1 as further evidence for the role of this gene in eye defects  including retinal coloboma  optic pallor  and glaucoma. We used Clustered Regularly Interspaced Short Palindromic Repeats CRISPR/Cas9 to make a stable Danio rerio model of crim1 deficiency  generating zebrafish that were homozygous for a 2 basepair deletion  c.339 340delCT p.Leu112Leufs*3  in crim1. Homozygous  crim1 /  larvae demonstrated smaller eyes and small and misshapen lenses compared to controls  but we did not observe colobomas. Bulk RNA Seq using dissected eyes from crim1 /  larvae and controls at 72 hpf showed significant downregulation of crim1 and chloride intracellular channel 4 clic4 and upregulation of fibroblast growth factor 1b fgf1b and complement component 1  q subcomponent c1q  amongst other dysregulated genes. Our work strengthens the association between haploinsufficiency for CRIM1 and eye defects and characterizes a stable model of crim1 loss of function for future research. Overall design: We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf. Wildtype and crim1 /  have two replicates.,,pubmed:40114969,,wildtype whole eyes  72 hpf  sample 2,GSM8794303,,source name:Whole eye|tissue:Whole eye|cell type:All|genotype:Wildtype|geo loc name:missing|collection date:missing,wildtype whole eyes  72 hpf  sample 2,The quality of raw single end reads of RNA Seq dataset was tested by FastQC http:// www.bioinformatics.babraham.ac.uk/projects/fastqc/ to evaluate the per base sequence quality  quality scores  sequence length distribution and overrepresented adapter/primer sequences.  post quality control with FastQC  data trimming and clipping was performed using BBDuk and aligned against the zebrafish reference genome GRCz11 using STAR. Alignment results were then assessed to check the quality with Qualimap. RSEM was used to quantify genes. Finally  DESeq2 was used to do the downstream analysis for differentially expressed genes. Statistically significant differentially expressed genes DEGs with log2 fold change greater than ±0.5 with an adjusted p value ≤0.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM ≥1 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script. Principal component analysis PCA was performed Assembly: GRCz11 Supplementary files format and content: Excel; Statistically significant differentially expressed genes DEGs with log2 fold change greater than ±0.5 with an adjusted p value ≤0.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM ≥1 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script.,Whole eye,,Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed ‘bulk’ RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation® RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5′ end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3′ end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.,,tissue:Whole eye|cell type:All|genotype:Wildtype,GSM8794303,GSM8794303: wildtype whole eyes  72 hpf  sample 2; Danio rerio; RNA Seq,GSM8794303 r1,GSM8794303,1,Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed 'bulk' RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation® RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5′ end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3′ end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP563805,,,crim1-ctr-2_S20_L003_R1_001.fastq.gz,fastq,2380780182.0,36072427.0,GSM8794303 r1,0:66,A:617266566;C:538355433;G:528639561;T:696293971;N:224651,66,,,,617266566,538355433,528639561,696293971,224651,SRX27670018,SRS24074873,,,Cincinnati Children's Hospital Medical Center,,,,,,,,,,,,B,,usable mapping rate,illumina,hiseq_era,unknown,random_priming,unknown,bulk,bulk,bulk,,United States,2025-02-13,Larval,Larval,Eye,Sensory System
34918,SRR32335089,SRX27670017,SRS24074872,SRP563805,PRJNA1223441,A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression ,GSE289562,Transcriptome Analysis,We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf.  This work will be published as Le et al.  2025: A zebrafish model of crim1 loss of function has small and misshapen lenses with dysregulated clic4 and fgf1b expression. Heterozygous deletions predicting haploinsufficiency for the Cysteine Rich Motor Neuron 1 CRIM1 gene have been identified in two families with macrophthalmia  colobomatous  with microcornea MACOM  an autosomal dominant trait. Crim1 encodes a type I transmembrane protein that is expressed at the cell membrane of lens epithelial and fiber cells at the stage of lens pit formation. Decreased Crim1 expression in the mouse reduced the number of lens epithelial cells and caused defective adhesion between lens epithelial cells and between the epithelial and fiber cells. We present three patients with heterozygous deletions and truncating variants predicted to result in haploinsufficiency for CRIM1 as further evidence for the role of this gene in eye defects  including retinal coloboma  optic pallor  and glaucoma. We used Clustered Regularly Interspaced Short Palindromic Repeats CRISPR/Cas9 to make a stable Danio rerio model of crim1 deficiency  generating zebrafish that were homozygous for a 2 basepair deletion  c.339 340delCT p.Leu112Leufs*3  in crim1. Homozygous  crim1 /  larvae demonstrated smaller eyes and small and misshapen lenses compared to controls  but we did not observe colobomas. Bulk RNA Seq using dissected eyes from crim1 /  larvae and controls at 72 hpf showed significant downregulation of crim1 and chloride intracellular channel 4 clic4 and upregulation of fibroblast growth factor 1b fgf1b and complement component 1  q subcomponent c1q  amongst other dysregulated genes. Our work strengthens the association between haploinsufficiency for CRIM1 and eye defects and characterizes a stable model of crim1 loss of function for future research. Overall design: We c ompared gene expression in dissected whole eyes from crim1 /  zebrafish compared to wildtype contros at 72 hpf. Wildtype and crim1 /  have two replicates.,,pubmed:40114969,,wildtype whole eyes  72 hpf  sample 1,GSM8794302,,source name:Whole eye|tissue:Whole eye|cell type:All|genotype:Wildtype|geo loc name:missing|collection date:missing,wildtype whole eyes  72 hpf  sample 1,The quality of raw single end reads of RNA Seq dataset was tested by FastQC http:// www.bioinformatics.babraham.ac.uk/projects/fastqc/ to evaluate the per base sequence quality  quality scores  sequence length distribution and overrepresented adapter/primer sequences.  post quality control with FastQC  data trimming and clipping was performed using BBDuk and aligned against the zebrafish reference genome GRCz11 using STAR. Alignment results were then assessed to check the quality with Qualimap. RSEM was used to quantify genes. Finally  DESeq2 was used to do the downstream analysis for differentially expressed genes. Statistically significant differentially expressed genes DEGs with log2 fold change greater than ±0.5 with an adjusted p value ≤0.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM ≥1 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script. Principal component analysis PCA was performed Assembly: GRCz11 Supplementary files format and content: Excel; Statistically significant differentially expressed genes DEGs with log2 fold change greater than ±0.5 with an adjusted p value ≤0.05 and the mean Fragments Per Kilobase of Exon Per Million Fragments Mapped FPKM ≥1 in all datasets were identified by comparing crim1 /  mutants with control datasets using an in house Python script.,Whole eye,,Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed ‘bulk’ RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation® RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5′ end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3′ end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.,,tissue:Whole eye|cell type:All|genotype:Wildtype,GSM8794302,GSM8794302: wildtype whole eyes  72 hpf  sample 1; Danio rerio; RNA Seq,GSM8794302 r1,GSM8794302,1,Whole eyes were dissected fromzebrafish at 72 hpf according to prior methods Krall et al.  2018 We performed 'bulk' RNA Seq experiments in dissected eyes from in crossed crim1 /  larvae and controls at 72 hpf according to prior methods Krall et al.  2018. RNA quality and quantity was reviewed with a bioanalyzer 2100 Bioanalyzer  Agilent and libraries were prepared with an Ovation® RNA Seq system V2 kit NuGEN. With this method  total RNA was reverse transcribed to synthesize first strand cDNA using a combination of random hexamers and a poly T chimeric primer Krall et al.  2018. The RNA template was then partially degraded by heating and the second strand cDNA was synthesized using DNA polymerase. The double stranded DNA was then amplified using single primer isothermal amplification SPIA. SPIA is a linear cDNA amplification process in which RNase H degrades RNA in DNA/RNA heteroduplex at the 5′ end of the double  stranded DNA  post which the SPIA primer binds to the cDNA and the polymerase starts replication at the 3′ end of the primer by displacement of the existing forward strand Krall et al.  2018. Random hexamers were then used to amplify the second strand cDNA linearly. Finally  libraries from the SPIA amplified cDNA were made using the Ultralow DR library kit NuGEN. The RNA Seq libraries were analyzed for quality and primer dimers by bioanalyzer and quantified by quantitative polymerase chain reaction qPCR prior to sequencing KAPA library quantification kit. High throughput sequencing was done using single end 50 lanes on a HiSeq 4000 instrument Illumina.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 4000,,SRP563805,,,crim1-ctr-1_S17_L003_R1_001.fastq.gz,fastq,3007758600.0,45572100.0,GSM8794302 r1,0:66,A:783035033;C:680405728;G:668894784;T:875142229;N:280826,66,,,,783035033,680405728,668894784,875142229,280826,SRX27670017,SRS24074872,,,Cincinnati Children's Hospital Medical Center,,,,,,,,,,,,B,,usable mapping rate,illumina,hiseq_era,unknown,random_priming,unknown,bulk,bulk,bulk,,United States,2025-02-13,Larval,Larval,Eye,Sensory System
36699,SRR835180,SRX271976,SRS416272,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,,rpeh 143h 31 436,GSM1129638,,tissue:retinal pigmented epithelium|hpf,rpeh 143h 31 436,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,retinal pigmented epithelium,,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,GSM1129638,GSM1129638: rpeh 143h 31 436; Danio rerio; RNA Seq,GSM1129638 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:GSM1129638,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,rpeh_143h_31_436_ATCT.fq.bz2,fastq,233585940.0,5561570.0,GSM1129638 r1,0:42,A:55598949;C:61313735;G:51248287;T:65240290;N:184679,42,,,,55598949,61313735,51248287,65240290,184679,SRX271976,SRS416272,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.81459,,0.17202,,0.85642,,0.45987,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Eye,Sensory System
36700,SRR835179,SRX271975,SRS416270,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,,rpeh 143h 31 351,GSM1129637,,tissue:retinal pigmented epithelium|hpf,rpeh 143h 31 351,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,retinal pigmented epithelium,,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,GSM1129637,GSM1129637: rpeh 143h 31 351; Danio rerio; RNA Seq,GSM1129637 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:GSM1129637,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,rpeh_143h_31_351_ATCT.fq.bz2,fastq,130461444.0,3623929.0,GSM1129637 r1,0:36,A:30448136;C:34618233;G:27756164;T:37637830;N:1081,36,,,,30448136,34618233,27756164,37637830,1081,SRX271975,SRS416270,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.80013,,0.18213,,0.85673,,0.46503,,36,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Eye,Sensory System
36701,SRR835178,SRX271974,SRS416271,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,,rpeh 86h 33 436,GSM1129636,,tissue:retinal pigmented epithelium|hpf,rpeh 86h 33 436,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,retinal pigmented epithelium,,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,GSM1129636,GSM1129636: rpeh 86h 33 436; Danio rerio; RNA Seq,GSM1129636 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:GSM1129636,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,rpeh_86h_33_436_CTTT.fq.bz2,fastq,375701970.0,8945285.0,GSM1129636 r1,0:42,A:79041659;C:105394908;G:87165145;T:103778792;N:321466,42,,,,79041659,105394908,87165145,103778792,321466,SRX271974,SRS416271,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.69127,,0.03078,,0.88016,,0.45532,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Eye,Sensory System
36702,SRR835177,SRX271973,SRS416269,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,,rpeh 86h 33 351,GSM1129635,,tissue:retinal pigmented epithelium|hpf,rpeh 86h 33 351,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,retinal pigmented epithelium,,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,GSM1129635,GSM1129635: rpeh 86h 33 351; Danio rerio; RNA Seq,GSM1129635 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:GSM1129635,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,rpeh_86h_33_351_CTTT.fq.bz2,fastq,94727196.0,2631311.0,GSM1129635 r1,0:36,A:19052917;C:27063976;G:21468497;T:27141011;N:795,36,,,,19052917,27063976,21468497,27141011,795,SRX271973,SRS416269,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.63527,,0.03789,,0.88118,,0.42915,,36,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Eye,Sensory System
36703,SRR835176,SRX271972,SRS416268,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,,rpeh 86h 32 436,GSM1129634,,tissue:retinal pigmented epithelium|hpf,rpeh 86h 32 436,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,retinal pigmented epithelium,,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,GSM1129634,GSM1129634: rpeh 86h 32 436; Danio rerio; RNA Seq,GSM1129634 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:GSM1129634,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,rpeh_86h_32_436_ACGT.fq.bz2,fastq,455528976.0,10845928.0,GSM1129634 r1,0:42,A:104114782;C:122028793;G:112429635;T:116603169;N:352597,42,,,,104114782,122028793,112429635,116603169,352597,SRX271972,SRS416268,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.80425,,0.07901,,0.83999,,0.53561,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Eye,Sensory System
36704,SRR835175,SRX271971,SRS416267,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,,rpeh 86h 32 351,GSM1129633,,tissue:retinal pigmented epithelium|hpf,rpeh 86h 32 351,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,retinal pigmented epithelium,,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,GSM1129633,GSM1129633: rpeh 86h 32 351; Danio rerio; RNA Seq,GSM1129633 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:GSM1129633,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,rpeh_86h_32_351_ACGT.fq.bz2,fastq,124319592.0,3453322.0,GSM1129633 r1,0:36,A:28020637;C:33641464;G:30211380;T:32445126;N:985,36,,,,28020637,33641464,30211380,32445126,985,SRX271971,SRS416267,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.80002,,0.09087,,0.83755,,0.53605,,36,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Eye,Sensory System
36705,SRR835174,SRX271970,SRS416266,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,,rpeleye 77h 48 440,GSM1129632,,tissue:retinal pigmented epithelium|hpf,rpeleye 77h 48 440,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,retinal pigmented epithelium,,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,GSM1129632,GSM1129632: rpeleye 77h 48 440; Danio rerio; RNA Seq,GSM1129632 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:GSM1129632,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,rpeleye_77h_48_440.fq.bz2,fastq,229484094.0,5463907.0,GSM1129632 r1,0:42,A:52877590;C:49045786;G:47421087;T:80125185;N:14446,42,,,,52877590,49045786,47421087,80125185,14446,SRX271970,SRS416266,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.8073,,0.19597,,0.84017,,0.51585,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Eye,Sensory System
36706,SRR835173,SRX271969,SRS416265,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,,rpeleye 77h 47 440,GSM1129631,,tissue:retinal pigmented epithelium|hpf,rpeleye 77h 47 440,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,retinal pigmented epithelium,,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,GSM1129631,GSM1129631: rpeleye 77h 47 440; Danio rerio; RNA Seq,GSM1129631 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:GSM1129631,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP021517,,,rpeleye_77h_47_440.fq.bz2,fastq,227857098.0,5425169.0,GSM1129631 r1,0:42,A:56239823;C:56488585;G:57018473;T:58097500;N:12717,42,,,,56239823,56488585,57018473,58097500,12717,SRX271969,SRS416265,SRA074390,GEO,"Rob Mitra, Genetics, Washington University",1,0.71632,,0.09237,,0.85602,,0.53769,,42,,B,,usable mapping rate,illumina,hiseq_era,3prime,poly_a,unknown,bulk,unknown,unknown,,United States,2013-04-25,Larval,Larval,Eye,Sensory System
40765,SRR3355805,SRX1689713,SRS1384139,SRP073099,PRJNA318016,Tet mediated DNA hydroxymethylation regulates retinal neurogenesis in zebrafish via cell extrinsic signaling pathways,GSE80134,Transcriptome Analysis,Using zebrafish tet2 / ;tet3 /  mutants  we identify functions for Tet enzymes and 5 hydroxymethylcytosine 5hmC in regulating gene expression and cell type specific differentiation during retinal development. Overall design: RNAseq from tet2 / ;tet3 /  mutant and sibling embryonic eye tissues dissected at 72hpf and 36hpf.,,pubmed:28926578,,sibling at 72hpf,GSM2113394,,source name:whole eye tissue|strain:TL|genotype/variation:sibling control|tissue:dissected eyes|age:72 hpf,sibling at 72hpf,base calling  demultiplexing done at the Genome Sequenicng and Analysis Facility  UT Austin fastq files quality checked using FastQC mapped using TopHat with annotated spliced junction gene counting and differentially expressed genes identified using Tuxedo Suite Trapnell et al.  2012 Genome build: GRCz10,whole eye tissue,none,whole eyes were dissected from fresh euthanized embryos. RNA immediately extracted using TriZol and QIAGEN RNeasy mini columns following manufacturer's protocol RNA samples were processed for RNAseq using Ambion poly A capture kit and NEB Next kit for cDNA synthesis and library prep.,zebrafish embryos raised under standard condition,strain:TL|genotype/variation:sibling control|tissue:dissected eyes|age:72 hpf,GSM2113394,GSM2113394: sibling at 72hpf; Danio rerio; RNA Seq,GSM2113394,,1,whole eyes were dissected from fresh euthanized embryos. RNA immediately extracted using TriZol and QIAGEN RNeasy mini columns following manufacturer's protocol RNA samples were processed for RNAseq using Ambion poly A capture kit and NEB Next kit for cDNA synthesis and library prep.,GEO Accession:GSM2113394,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP073099,,,tet23_WT_GTCCGC_L001_R1_001.fastq.gz tet23_WT_GTCCGC_L001_R2_001.fastq.gz,fastq fastq,2760449832.0,10954166.0,GSM2113394 r1,0:126 1:126,A:749168042;C:630997286;G:640050974;T:738938277;N:1295253,126,126,,,749168042,630997286,640050974,738938277,1295253,SRX1689713,SRS1384139,SRA409011,GEO,"Gross, Ophthalmology, University of Pittsburgh",2,0.94057,0.93996,0.11845,0.11794,0.70088,0.70193,0.45873,0.45468,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nebnext,bulk,unknown,unknown,,United States,2016-04-11,Larval,Larval,Eye,Sensory System
40766,SRR3355806,SRX1689713,SRS1384139,SRP073099,PRJNA318016,Tet mediated DNA hydroxymethylation regulates retinal neurogenesis in zebrafish via cell extrinsic signaling pathways,GSE80134,Transcriptome Analysis,Using zebrafish tet2 / ;tet3 /  mutants  we identify functions for Tet enzymes and 5 hydroxymethylcytosine 5hmC in regulating gene expression and cell type specific differentiation during retinal development. Overall design: RNAseq from tet2 / ;tet3 /  mutant and sibling embryonic eye tissues dissected at 72hpf and 36hpf.,,pubmed:28926578,,sibling at 72hpf,GSM2113394,,source name:whole eye tissue|strain:TL|genotype/variation:sibling control|tissue:dissected eyes|age:72 hpf,sibling at 72hpf,base calling  demultiplexing done at the Genome Sequenicng and Analysis Facility  UT Austin fastq files quality checked using FastQC mapped using TopHat with annotated spliced junction gene counting and differentially expressed genes identified using Tuxedo Suite Trapnell et al.  2012 Genome build: GRCz10,whole eye tissue,none,whole eyes were dissected from fresh euthanized embryos. RNA immediately extracted using TriZol and QIAGEN RNeasy mini columns following manufacturer's protocol RNA samples were processed for RNAseq using Ambion poly A capture kit and NEB Next kit for cDNA synthesis and library prep.,zebrafish embryos raised under standard condition,strain:TL|genotype/variation:sibling control|tissue:dissected eyes|age:72 hpf,GSM2113394,GSM2113394: sibling at 72hpf; Danio rerio; RNA Seq,GSM2113394,,1,whole eyes were dissected from fresh euthanized embryos. RNA immediately extracted using TriZol and QIAGEN RNeasy mini columns following manufacturer's protocol RNA samples were processed for RNAseq using Ambion poly A capture kit and NEB Next kit for cDNA synthesis and library prep.,GEO Accession:GSM2113394,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP073099,,,tet23_WT_GTCCGC_L002_R1_001.fastq.gz tet23_WT_GTCCGC_L002_R2_001.fastq.gz,fastq fastq,2656145016.0,10540258.0,GSM2113394 r2,0:126 1:126,A:720508816;C:607627764;G:616329145;T:710634294;N:1044997,126,126,,,720508816,607627764,616329145,710634294,1044997,SRX1689713,SRS1384139,SRA409011,GEO,"Gross, Ophthalmology, University of Pittsburgh",2,0.94056,0.94201,0.1166,0.1169,0.70067,0.70161,0.45652,0.45467,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nebnext,bulk,unknown,unknown,,United States,2016-04-11,Larval,Larval,Eye,Sensory System
40767,SRR3355807,SRX1689713,SRS1384139,SRP073099,PRJNA318016,Tet mediated DNA hydroxymethylation regulates retinal neurogenesis in zebrafish via cell extrinsic signaling pathways,GSE80134,Transcriptome Analysis,Using zebrafish tet2 / ;tet3 /  mutants  we identify functions for Tet enzymes and 5 hydroxymethylcytosine 5hmC in regulating gene expression and cell type specific differentiation during retinal development. Overall design: RNAseq from tet2 / ;tet3 /  mutant and sibling embryonic eye tissues dissected at 72hpf and 36hpf.,,pubmed:28926578,,sibling at 72hpf,GSM2113394,,source name:whole eye tissue|strain:TL|genotype/variation:sibling control|tissue:dissected eyes|age:72 hpf,sibling at 72hpf,base calling  demultiplexing done at the Genome Sequenicng and Analysis Facility  UT Austin fastq files quality checked using FastQC mapped using TopHat with annotated spliced junction gene counting and differentially expressed genes identified using Tuxedo Suite Trapnell et al.  2012 Genome build: GRCz10,whole eye tissue,none,whole eyes were dissected from fresh euthanized embryos. RNA immediately extracted using TriZol and QIAGEN RNeasy mini columns following manufacturer's protocol RNA samples were processed for RNAseq using Ambion poly A capture kit and NEB Next kit for cDNA synthesis and library prep.,zebrafish embryos raised under standard condition,strain:TL|genotype/variation:sibling control|tissue:dissected eyes|age:72 hpf,GSM2113394,GSM2113394: sibling at 72hpf; Danio rerio; RNA Seq,GSM2113394,,1,whole eyes were dissected from fresh euthanized embryos. RNA immediately extracted using TriZol and QIAGEN RNeasy mini columns following manufacturer's protocol RNA samples were processed for RNAseq using Ambion poly A capture kit and NEB Next kit for cDNA synthesis and library prep.,GEO Accession:GSM2113394,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP073099,,,tet23_WT_GTCCGC_L003_R1_001.fastq.gz tet23_WT_GTCCGC_L003_R2_001.fastq.gz,fastq fastq,2328068232.0,9238366.0,GSM2113394 r3,0:126 1:126,A:631534603;C:532516326;G:540288768;T:622815064;N:913471,126,126,,,631534603,532516326,540288768,622815064,913471,SRX1689713,SRS1384139,SRA409011,GEO,"Gross, Ophthalmology, University of Pittsburgh",2,0.94072,0.94244,0.11762,0.11706,0.69984,0.70128,0.45536,0.45595,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nebnext,bulk,unknown,unknown,,United States,2016-04-11,Larval,Larval,Eye,Sensory System
40768,SRR3355808,SRX1689713,SRS1384139,SRP073099,PRJNA318016,Tet mediated DNA hydroxymethylation regulates retinal neurogenesis in zebrafish via cell extrinsic signaling pathways,GSE80134,Transcriptome Analysis,Using zebrafish tet2 / ;tet3 /  mutants  we identify functions for Tet enzymes and 5 hydroxymethylcytosine 5hmC in regulating gene expression and cell type specific differentiation during retinal development. Overall design: RNAseq from tet2 / ;tet3 /  mutant and sibling embryonic eye tissues dissected at 72hpf and 36hpf.,,pubmed:28926578,,sibling at 72hpf,GSM2113394,,source name:whole eye tissue|strain:TL|genotype/variation:sibling control|tissue:dissected eyes|age:72 hpf,sibling at 72hpf,base calling  demultiplexing done at the Genome Sequenicng and Analysis Facility  UT Austin fastq files quality checked using FastQC mapped using TopHat with annotated spliced junction gene counting and differentially expressed genes identified using Tuxedo Suite Trapnell et al.  2012 Genome build: GRCz10,whole eye tissue,none,whole eyes were dissected from fresh euthanized embryos. RNA immediately extracted using TriZol and QIAGEN RNeasy mini columns following manufacturer's protocol RNA samples were processed for RNAseq using Ambion poly A capture kit and NEB Next kit for cDNA synthesis and library prep.,zebrafish embryos raised under standard condition,strain:TL|genotype/variation:sibling control|tissue:dissected eyes|age:72 hpf,GSM2113394,GSM2113394: sibling at 72hpf; Danio rerio; RNA Seq,GSM2113394,,1,whole eyes were dissected from fresh euthanized embryos. RNA immediately extracted using TriZol and QIAGEN RNeasy mini columns following manufacturer's protocol RNA samples were processed for RNAseq using Ambion poly A capture kit and NEB Next kit for cDNA synthesis and library prep.,GEO Accession:GSM2113394,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP073099,,,tet23_WT_GTCCGC_L004_R1_001.fastq.gz tet23_WT_GTCCGC_L004_R2_001.fastq.gz,fastq fastq,1947086064.0,7726532.0,GSM2113394 r4,0:126 1:126,A:527512520;C:446273569;G:452903888;T:520069256;N:326831,126,126,,,527512520,446273569,452903888,520069256,326831,SRX1689713,SRS1384139,SRA409011,GEO,"Gross, Ophthalmology, University of Pittsburgh",2,0.94067,0.94272,0.11804,0.11769,0.7011,0.70197,0.45353,0.45327,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nebnext,bulk,unknown,unknown,,United States,2016-04-11,Larval,Larval,Eye,Sensory System
40769,SRR3355801,SRX1689712,SRS1384140,SRP073099,PRJNA318016,Tet mediated DNA hydroxymethylation regulates retinal neurogenesis in zebrafish via cell extrinsic signaling pathways,GSE80134,Transcriptome Analysis,Using zebrafish tet2 / ;tet3 /  mutants  we identify functions for Tet enzymes and 5 hydroxymethylcytosine 5hmC in regulating gene expression and cell type specific differentiation during retinal development. Overall design: RNAseq from tet2 / ;tet3 /  mutant and sibling embryonic eye tissues dissected at 72hpf and 36hpf.,,pubmed:28926578,,tet2 / ;tet3 /  mutants at 72hpf,GSM2113393,,source name:whole eye tissue|strain:TL|genotype/variation:tet2 / ;tet3 /  mutants|tissue:dissected eyes|age:72 hpf,tet2 / ;tet3 /  mutants at 72hpf,base calling  demultiplexing done at the Genome Sequenicng and Analysis Facility  UT Austin fastq files quality checked using FastQC mapped using TopHat with annotated spliced junction gene counting and differentially expressed genes identified using Tuxedo Suite Trapnell et al.  2012 Genome build: GRCz10,whole eye tissue,none,whole eyes were dissected from fresh euthanized embryos. RNA immediately extracted using TriZol and QIAGEN RNeasy mini columns following manufacturer's protocol RNA samples were processed for RNAseq using Ambion poly A capture kit and NEB Next kit for cDNA synthesis and library prep.,zebrafish embryos raised under standard condition,strain:TL|genotype/variation:tet2 / ;tet3 /  mutants|tissue:dissected eyes|age:72 hpf,GSM2113393,GSM2113393: tet2 / ;tet3 /  mutants at 72hpf; Danio rerio; RNA Seq,GSM2113393,,1,whole eyes were dissected from fresh euthanized embryos. RNA immediately extracted using TriZol and QIAGEN RNeasy mini columns following manufacturer's protocol RNA samples were processed for RNAseq using Ambion poly A capture kit and NEB Next kit for cDNA synthesis and library prep.,GEO Accession:GSM2113393,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP073099,,,tet23_MU_GTAGAG_L001_R1_001.fastq.gz tet23_MU_GTAGAG_L001_R2_001.fastq.gz,fastq fastq,1912689576.0,7590038.0,GSM2113393 r1,0:126 1:126,A:512456960;C:443202950;G:451169263;T:504937998;N:922405,126,126,,,512456960,443202950,451169263,504937998,922405,SRX1689712,SRS1384140,SRA409011,GEO,"Gross, Ophthalmology, University of Pittsburgh",2,0.94424,0.94429,0.10083,0.10068,0.69666,0.69998,0.46271,0.46191,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nebnext,bulk,unknown,unknown,,United States,2016-04-11,Larval,Larval,Eye,Sensory System
40770,SRR3355802,SRX1689712,SRS1384140,SRP073099,PRJNA318016,Tet mediated DNA hydroxymethylation regulates retinal neurogenesis in zebrafish via cell extrinsic signaling pathways,GSE80134,Transcriptome Analysis,Using zebrafish tet2 / ;tet3 /  mutants  we identify functions for Tet enzymes and 5 hydroxymethylcytosine 5hmC in regulating gene expression and cell type specific differentiation during retinal development. Overall design: RNAseq from tet2 / ;tet3 /  mutant and sibling embryonic eye tissues dissected at 72hpf and 36hpf.,,pubmed:28926578,,tet2 / ;tet3 /  mutants at 72hpf,GSM2113393,,source name:whole eye tissue|strain:TL|genotype/variation:tet2 / ;tet3 /  mutants|tissue:dissected eyes|age:72 hpf,tet2 / ;tet3 /  mutants at 72hpf,base calling  demultiplexing done at the Genome Sequenicng and Analysis Facility  UT Austin fastq files quality checked using FastQC mapped using TopHat with annotated spliced junction gene counting and differentially expressed genes identified using Tuxedo Suite Trapnell et al.  2012 Genome build: GRCz10,whole eye tissue,none,whole eyes were dissected from fresh euthanized embryos. RNA immediately extracted using TriZol and QIAGEN RNeasy mini columns following manufacturer's protocol RNA samples were processed for RNAseq using Ambion poly A capture kit and NEB Next kit for cDNA synthesis and library prep.,zebrafish embryos raised under standard condition,strain:TL|genotype/variation:tet2 / ;tet3 /  mutants|tissue:dissected eyes|age:72 hpf,GSM2113393,GSM2113393: tet2 / ;tet3 /  mutants at 72hpf; Danio rerio; RNA Seq,GSM2113393,,1,whole eyes were dissected from fresh euthanized embryos. RNA immediately extracted using TriZol and QIAGEN RNeasy mini columns following manufacturer's protocol RNA samples were processed for RNAseq using Ambion poly A capture kit and NEB Next kit for cDNA synthesis and library prep.,GEO Accession:GSM2113393,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP073099,,,tet23_MU_GTAGAG_L002_R1_001.fastq.gz tet23_MU_GTAGAG_L002_R2_001.fastq.gz,fastq fastq,1798682760.0,7137630.0,GSM2113393 r2,0:126 1:126,A:481684544;C:417096435;G:424517786;T:474657513;N:726482,126,126,,,481684544,417096435,424517786,474657513,726482,SRX1689712,SRS1384140,SRA409011,GEO,"Gross, Ophthalmology, University of Pittsburgh",2,0.94542,0.94741,0.10191,0.10175,0.69877,0.70043,0.46256,0.45996,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nebnext,bulk,unknown,unknown,,United States,2016-04-11,Larval,Larval,Eye,Sensory System
40771,SRR3355803,SRX1689712,SRS1384140,SRP073099,PRJNA318016,Tet mediated DNA hydroxymethylation regulates retinal neurogenesis in zebrafish via cell extrinsic signaling pathways,GSE80134,Transcriptome Analysis,Using zebrafish tet2 / ;tet3 /  mutants  we identify functions for Tet enzymes and 5 hydroxymethylcytosine 5hmC in regulating gene expression and cell type specific differentiation during retinal development. Overall design: RNAseq from tet2 / ;tet3 /  mutant and sibling embryonic eye tissues dissected at 72hpf and 36hpf.,,pubmed:28926578,,tet2 / ;tet3 /  mutants at 72hpf,GSM2113393,,source name:whole eye tissue|strain:TL|genotype/variation:tet2 / ;tet3 /  mutants|tissue:dissected eyes|age:72 hpf,tet2 / ;tet3 /  mutants at 72hpf,base calling  demultiplexing done at the Genome Sequenicng and Analysis Facility  UT Austin fastq files quality checked using FastQC mapped using TopHat with annotated spliced junction gene counting and differentially expressed genes identified using Tuxedo Suite Trapnell et al.  2012 Genome build: GRCz10,whole eye tissue,none,whole eyes were dissected from fresh euthanized embryos. RNA immediately extracted using TriZol and QIAGEN RNeasy mini columns following manufacturer's protocol RNA samples were processed for RNAseq using Ambion poly A capture kit and NEB Next kit for cDNA synthesis and library prep.,zebrafish embryos raised under standard condition,strain:TL|genotype/variation:tet2 / ;tet3 /  mutants|tissue:dissected eyes|age:72 hpf,GSM2113393,GSM2113393: tet2 / ;tet3 /  mutants at 72hpf; Danio rerio; RNA Seq,GSM2113393,,1,whole eyes were dissected from fresh euthanized embryos. RNA immediately extracted using TriZol and QIAGEN RNeasy mini columns following manufacturer's protocol RNA samples were processed for RNAseq using Ambion poly A capture kit and NEB Next kit for cDNA synthesis and library prep.,GEO Accession:GSM2113393,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP073099,,,tet23_MU_GTAGAG_L003_R1_001.fastq.gz tet23_MU_GTAGAG_L003_R2_001.fastq.gz,fastq fastq,1690181892.0,6707071.0,GSM2113393 r3,0:126 1:126,A:452345344;C:392188748;G:399266597;T:445684554;N:696649,126,126,,,452345344,392188748,399266597,445684554,696649,SRX1689712,SRS1384140,SRA409011,GEO,"Gross, Ophthalmology, University of Pittsburgh",2,0.9457,0.94788,0.10141,0.10082,0.69828,0.69962,0.46578,0.46406,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nebnext,bulk,unknown,unknown,,United States,2016-04-11,Larval,Larval,Eye,Sensory System
40772,SRR3355804,SRX1689712,SRS1384140,SRP073099,PRJNA318016,Tet mediated DNA hydroxymethylation regulates retinal neurogenesis in zebrafish via cell extrinsic signaling pathways,GSE80134,Transcriptome Analysis,Using zebrafish tet2 / ;tet3 /  mutants  we identify functions for Tet enzymes and 5 hydroxymethylcytosine 5hmC in regulating gene expression and cell type specific differentiation during retinal development. Overall design: RNAseq from tet2 / ;tet3 /  mutant and sibling embryonic eye tissues dissected at 72hpf and 36hpf.,,pubmed:28926578,,tet2 / ;tet3 /  mutants at 72hpf,GSM2113393,,source name:whole eye tissue|strain:TL|genotype/variation:tet2 / ;tet3 /  mutants|tissue:dissected eyes|age:72 hpf,tet2 / ;tet3 /  mutants at 72hpf,base calling  demultiplexing done at the Genome Sequenicng and Analysis Facility  UT Austin fastq files quality checked using FastQC mapped using TopHat with annotated spliced junction gene counting and differentially expressed genes identified using Tuxedo Suite Trapnell et al.  2012 Genome build: GRCz10,whole eye tissue,none,whole eyes were dissected from fresh euthanized embryos. RNA immediately extracted using TriZol and QIAGEN RNeasy mini columns following manufacturer's protocol RNA samples were processed for RNAseq using Ambion poly A capture kit and NEB Next kit for cDNA synthesis and library prep.,zebrafish embryos raised under standard condition,strain:TL|genotype/variation:tet2 / ;tet3 /  mutants|tissue:dissected eyes|age:72 hpf,GSM2113393,GSM2113393: tet2 / ;tet3 /  mutants at 72hpf; Danio rerio; RNA Seq,GSM2113393,,1,whole eyes were dissected from fresh euthanized embryos. RNA immediately extracted using TriZol and QIAGEN RNeasy mini columns following manufacturer's protocol RNA samples were processed for RNAseq using Ambion poly A capture kit and NEB Next kit for cDNA synthesis and library prep.,GEO Accession:GSM2113393,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP073099,,,tet23_MU_GTAGAG_L004_R1_001.fastq.gz tet23_MU_GTAGAG_L004_R2_001.fastq.gz,fastq fastq,1571944752.0,6237876.0,GSM2113393 r4,0:126 1:126,A:420346043;C:365280582;G:371972942;T:414077272;N:267913,126,126,,,420346043,365280582,371972942,414077272,267913,SRX1689712,SRS1384140,SRA409011,GEO,"Gross, Ophthalmology, University of Pittsburgh",2,0.94661,0.94872,0.1011,0.10112,0.69627,0.69753,0.45952,0.46497,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nebnext,bulk,unknown,unknown,,United States,2016-04-11,Larval,Larval,Eye,Sensory System
42018,SRR5382066,SRX2677147,SRS2075568,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,NO shh 13,GSM2552024,,tissue:whole larval eyes|genotype:wild type|age:5 dpf,NO shh 13,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:wild type|age:5 dpf,GSM2552024,GSM2552024: NO shh 13; Danio rerio; RNA Seq,GSM2552024,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552024,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L6_HKZEBxagRABIRAAPEI-214_1.fq.gz,fastq,843818269.0,17220781.0,GSM2552024 r1,0:49 1:0,A:221838981;C:201486730;G:194808523;T:225676801;N:7234,49,0,,,221838981,201486730,194808523,225676801,7234,SRX2677147,SRS2075568,SRA549807,GEO,Medical College of Wisconsin,1,0.90043,,0.1077,,0.68897,,0.40147,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42019,SRR5382067,SRX2677147,SRS2075568,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,NO shh 13,GSM2552024,,tissue:whole larval eyes|genotype:wild type|age:5 dpf,NO shh 13,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:wild type|age:5 dpf,GSM2552024,GSM2552024: NO shh 13; Danio rerio; RNA Seq,GSM2552024,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552024,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L7_HKZEBxagRABIRAAPEI-214_1.fq.gz,fastq,829961510.0,16937990.0,GSM2552024 r2,0:49 1:0,A:218002306;C:198331442;G:191775017;T:221844452;N:8293,49,0,,,218002306,198331442,191775017,221844452,8293,SRX2677147,SRS2075568,SRA549807,GEO,Medical College of Wisconsin,1,0.90751,,0.10841,,0.6882,,0.40509,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42020,SRR5382064,SRX2677146,SRS2075567,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,NO shh 12,GSM2552023,,tissue:whole larval eyes|genotype:wild type|age:5 dpf,NO shh 12,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:wild type|age:5 dpf,GSM2552023,GSM2552023: NO shh 12; Danio rerio; RNA Seq,GSM2552023,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552023,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L6_HKZEBxagRABGRAAPEI-212_1.fq.gz,fastq,840785561.0,17158889.0,GSM2552023 r1,0:49 1:0,A:221201132;C:200524999;G:193274512;T:225777636;N:7282,49,0,,,221201132,200524999,193274512,225777636,7282,SRX2677146,SRS2075567,SRA549807,GEO,Medical College of Wisconsin,1,0.90205,,0.11292,,0.68759,,0.44522,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42021,SRR5382065,SRX2677146,SRS2075567,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,NO shh 12,GSM2552023,,tissue:whole larval eyes|genotype:wild type|age:5 dpf,NO shh 12,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:wild type|age:5 dpf,GSM2552023,GSM2552023: NO shh 12; Danio rerio; RNA Seq,GSM2552023,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552023,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L7_HKZEBxagRABGRAAPEI-212_1.fq.gz,fastq,834811089.0,17036961.0,GSM2552023 r2,0:49 1:0,A:219382137;C:199232543;G:192142641;T:224045481;N:8287,49,0,,,219382137,199232543,192142641,224045481,8287,SRX2677146,SRS2075567,SRA549807,GEO,Medical College of Wisconsin,1,0.90899,,0.11358,,0.68809,,0.43149,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42022,SRR5382062,SRX2677145,SRS2075566,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,NO shh 11,GSM2552022,,tissue:whole larval eyes|genotype:wild type|age:5 dpf,NO shh 11,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:wild type|age:5 dpf,GSM2552022,GSM2552022: NO shh 11; Danio rerio; RNA Seq,GSM2552022,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552022,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L6_HKZEBxagRABERAAPEI-210_1.fq.gz,fastq,904157212.0,18452188.0,GSM2552022 r1,0:49 1:0,A:236992487;C:215051901;G:210839328;T:241266600;N:6896,49,0,,,236992487,215051901,210839328,241266600,6896,SRX2677145,SRS2075566,SRA549807,GEO,Medical College of Wisconsin,1,0.89893,,0.10891,,0.68465,,0.44598,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42023,SRR5382063,SRX2677145,SRS2075566,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,NO shh 11,GSM2552022,,tissue:whole larval eyes|genotype:wild type|age:5 dpf,NO shh 11,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:wild type|age:5 dpf,GSM2552022,GSM2552022: NO shh 11; Danio rerio; RNA Seq,GSM2552022,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552022,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L7_HKZEBxagRABERAAPEI-210_1.fq.gz,fastq,896869197.0,18303453.0,GSM2552022 r2,0:49 1:0,A:234904626;C:213413392;G:209296940;T:239246543;N:7696,49,0,,,234904626,213413392,209296940,239246543,7696,SRX2677145,SRS2075566,SRA549807,GEO,Medical College of Wisconsin,1,0.9066,,0.1098,,0.68444,,0.45516,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42024,SRR5382060,SRX2677144,SRS2075565,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,shh 13,GSM2552021,,tissue:whole larval eyes|genotype:Transgenic hsp70l:shha eGFP|age:5 dpf,shh 13,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:Transgenic hsp70l:shha eGFP|age:5 dpf,GSM2552021,GSM2552021: shh 13; Danio rerio; RNA Seq,GSM2552021,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552021,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L6_HKZEBxagRABHRAAPEI-213_1.fq.gz,fastq,780154725.0,15921525.0,GSM2552021 r1,0:49 1:0,A:204493928;C:186497801;G:181452727;T:207703532;N:6737,49,0,,,204493928,186497801,181452727,207703532,6737,SRX2677144,SRS2075565,SRA549807,GEO,Medical College of Wisconsin,1,0.91024,,0.07095,,0.72874,,0.42618,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42025,SRR5382061,SRX2677144,SRS2075565,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,shh 13,GSM2552021,,tissue:whole larval eyes|genotype:Transgenic hsp70l:shha eGFP|age:5 dpf,shh 13,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:Transgenic hsp70l:shha eGFP|age:5 dpf,GSM2552021,GSM2552021: shh 13; Danio rerio; RNA Seq,GSM2552021,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552021,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L7_HKZEBxagRABHRAAPEI-213_1.fq.gz,fastq,723588096.0,14767104.0,GSM2552021 r2,0:49 1:0,A:189438629;C:173078510;G:168506893;T:192556891;N:7173,49,0,,,189438629,173078510,168506893,192556891,7173,SRX2677144,SRS2075565,SRA549807,GEO,Medical College of Wisconsin,1,0.91641,,0.07158,,0.7292,,0.41626,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42026,SRR5382058,SRX2677143,SRS2075564,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,shh 12,GSM2552020,,tissue:whole larval eyes|genotype:Transgenic hsp70l:shha eGFP|age:5 dpf,shh 12,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:Transgenic hsp70l:shha eGFP|age:5 dpf,GSM2552020,GSM2552020: shh 12; Danio rerio; RNA Seq,GSM2552020,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552020,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L6_HKZEBxagRABFRAAPEI-211_1.fq.gz,fastq,767433100.0,15661900.0,GSM2552020 r1,0:49 1:0,A:201134121;C:184183797;G:177805845;T:204302839;N:6498,49,0,,,201134121,184183797,177805845,204302839,6498,SRX2677143,SRS2075564,SRA549807,GEO,Medical College of Wisconsin,1,0.90254,,0.10915,,0.69029,,0.43993,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42027,SRR5382059,SRX2677143,SRS2075564,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,shh 12,GSM2552020,,tissue:whole larval eyes|genotype:Transgenic hsp70l:shha eGFP|age:5 dpf,shh 12,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:Transgenic hsp70l:shha eGFP|age:5 dpf,GSM2552020,GSM2552020: shh 12; Danio rerio; RNA Seq,GSM2552020,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552020,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L7_HKZEBxagRABFRAAPEI-211_1.fq.gz,fastq,748240290.0,15270210.0,GSM2552020 r2,0:49 1:0,A:195869449;C:179706491;G:173543679;T:199113253;N:7418,49,0,,,195869449,179706491,173543679,199113253,7418,SRX2677143,SRS2075564,SRA549807,GEO,Medical College of Wisconsin,1,0.90915,,0.11014,,0.68929,,0.42638,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42028,SRR5382056,SRX2677142,SRS2075563,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,shh 11,GSM2552019,,tissue:whole larval eyes|genotype:Transgenic hsp70l:shha eGFP|age:5 dpf,shh 11,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:Transgenic hsp70l:shha eGFP|age:5 dpf,GSM2552019,GSM2552019: shh 11; Danio rerio; RNA Seq,GSM2552019,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552019,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L6_HKZEBxagRABDRAAPEI-209_1.fq.gz,fastq,735291942.0,15005958.0,GSM2552019 r1,0:49 1:0,A:191931154;C:176849705;G:171335621;T:195169335;N:6127,49,0,,,191931154,176849705,171335621,195169335,6127,SRX2677142,SRS2075563,SRA549807,GEO,Medical College of Wisconsin,1,0.89997,,0.10193,,0.68771,,0.42739,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42029,SRR5382057,SRX2677142,SRS2075563,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,shh 11,GSM2552019,,tissue:whole larval eyes|genotype:Transgenic hsp70l:shha eGFP|age:5 dpf,shh 11,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:Transgenic hsp70l:shha eGFP|age:5 dpf,GSM2552019,GSM2552019: shh 11; Danio rerio; RNA Seq,GSM2552019,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552019,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L7_HKZEBxagRABDRAAPEI-209_1.fq.gz,fastq,738096016.0,15063184.0,GSM2552019 r2,0:49 1:0,A:192422803;C:177645752;G:172165273;T:195854853;N:7335,49,0,,,192422803,177645752,172165273,195854853,7335,SRX2677142,SRS2075563,SRA549807,GEO,Medical College of Wisconsin,1,0.90699,,0.1029,,0.68609,,0.4169,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42030,SRR5382054,SRX2677141,SRS2075562,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,NO bmp4 13,GSM2552018,,tissue:whole larval eyes|genotype:wild type|age:5 dpf,NO bmp4 13,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:wild type|age:5 dpf,GSM2552018,GSM2552018: NO bmp4 13; Danio rerio; RNA Seq,GSM2552018,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552018,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L6_HKZEBxagRABCRAAPEI-208_1.fq.gz,fastq,821111277.0,16757373.0,GSM2552018 r1,0:49 1:0,A:214147667;C:197777522;G:191357044;T:217822321;N:6723,49,0,,,214147667,197777522,191357044,217822321,6723,SRX2677141,SRS2075562,SRA549807,GEO,Medical College of Wisconsin,1,0.90103,,0.09455,,0.69098,,0.43034,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42031,SRR5382055,SRX2677141,SRS2075562,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,NO bmp4 13,GSM2552018,,tissue:whole larval eyes|genotype:wild type|age:5 dpf,NO bmp4 13,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:wild type|age:5 dpf,GSM2552018,GSM2552018: NO bmp4 13; Danio rerio; RNA Seq,GSM2552018,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552018,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L7_HKZEBxagRABCRAAPEI-208_1.fq.gz,fastq,858019596.0,17510604.0,GSM2552018 r2,0:49 1:0,A:223513905;C:206829946;G:200141599;T:227525603;N:8543,49,0,,,223513905,206829946,200141599,227525603,8543,SRX2677141,SRS2075562,SRA549807,GEO,Medical College of Wisconsin,1,0.90579,,0.09535,,0.68968,,0.43592,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42032,SRR5382052,SRX2677140,SRS2075561,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,NO bmp4 12,GSM2552017,,tissue:whole larval eyes|genotype:wild type|age:5 dpf,NO bmp4 12,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:wild type|age:5 dpf,GSM2552017,GSM2552017: NO bmp4 12; Danio rerio; RNA Seq,GSM2552017,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552017,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L6_HKZEBxagRABARAAPEI-206_1.fq.gz,fastq,893675524.0,18238276.0,GSM2552017 r1,0:49 1:0,A:233295131;C:215015502;G:208033601;T:237323630;N:7660,49,0,,,233295131,215015502,208033601,237323630,7660,SRX2677140,SRS2075561,SRA549807,GEO,Medical College of Wisconsin,1,0.89549,,0.10462,,0.68716,,0.44466,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42033,SRR5382053,SRX2677140,SRS2075561,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,NO bmp4 12,GSM2552017,,tissue:whole larval eyes|genotype:wild type|age:5 dpf,NO bmp4 12,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:wild type|age:5 dpf,GSM2552017,GSM2552017: NO bmp4 12; Danio rerio; RNA Seq,GSM2552017,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552017,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L7_HKZEBxagRABARAAPEI-206_1.fq.gz,fastq,897380218.0,18313882.0,GSM2552017 r2,0:49 1:0,A:234059121;C:216066812;G:208996578;T:238249498;N:8209,49,0,,,234059121,216066812,208996578,238249498,8209,SRX2677140,SRS2075561,SRA549807,GEO,Medical College of Wisconsin,1,0.90226,,0.10464,,0.68708,,0.43834,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42034,SRR5382050,SRX2677139,SRS2075560,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,NO bmp4 11,GSM2552016,,tissue:whole larval eyes|genotype:wild type|age:5 dpf,NO bmp4 11,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:wild type|age:5 dpf,GSM2552016,GSM2552016: NO bmp4 11; Danio rerio; RNA Seq,GSM2552016,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552016,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L6_HKZEBxagRAAYRAAPEI-203_1.fq.gz,fastq,761586714.0,15542586.0,GSM2552016 r1,0:49 1:0,A:199500708;C:182759688;G:176315916;T:203003924;N:6478,49,0,,,199500708,182759688,176315916,203003924,6478,SRX2677139,SRS2075560,SRA549807,GEO,Medical College of Wisconsin,1,0.89716,,0.10544,,0.6871,,0.43385,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42035,SRR5382051,SRX2677139,SRS2075560,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,NO bmp4 11,GSM2552016,,tissue:whole larval eyes|genotype:wild type|age:5 dpf,NO bmp4 11,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:wild type|age:5 dpf,GSM2552016,GSM2552016: NO bmp4 11; Danio rerio; RNA Seq,GSM2552016,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552016,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L7_HKZEBxagRAAYRAAPEI-203_1.fq.gz,fastq,777977998.0,15877102.0,GSM2552016 r2,0:49 1:0,A:203591828;C:186858026;G:180247771;T:207272648;N:7725,49,0,,,203591828,186858026,180247771,207272648,7725,SRX2677139,SRS2075560,SRA549807,GEO,Medical College of Wisconsin,1,0.90386,,0.10814,,0.68728,,0.44655,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42036,SRR5382048,SRX2677138,SRS2075559,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,bmp4 13,GSM2552015,,tissue:whole larval eyes|genotype:Transgenic hsp70l:eGFP bmp4|age:5 dpf,bmp4 13,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:Transgenic hsp70l:eGFP bmp4|age:5 dpf,GSM2552015,GSM2552015: bmp4 13; Danio rerio; RNA Seq,GSM2552015,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552015,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L6_HKZEBxagRABBRAAPEI-207_1.fq.gz,fastq,771609076.0,15747124.0,GSM2552015 r1,0:49 1:0,A:201323965;C:185589440;G:180186502;T:204502871;N:6298,49,0,,,201323965,185589440,180186502,204502871,6298,SRX2677138,SRS2075559,SRA549807,GEO,Medical College of Wisconsin,1,0.89654,,0.09972,,0.68921,,0.43468,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42037,SRR5382049,SRX2677138,SRS2075559,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,bmp4 13,GSM2552015,,tissue:whole larval eyes|genotype:Transgenic hsp70l:eGFP bmp4|age:5 dpf,bmp4 13,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:Transgenic hsp70l:eGFP bmp4|age:5 dpf,GSM2552015,GSM2552015: bmp4 13; Danio rerio; RNA Seq,GSM2552015,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552015,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L7_HKZEBxagRABBRAAPEI-207_1.fq.gz,fastq,770419650.0,15722850.0,GSM2552015 r2,0:49 1:0,A:200771257;C:185509037;G:180033647;T:204098225;N:7484,49,0,,,200771257,185509037,180033647,204098225,7484,SRX2677138,SRS2075559,SRA549807,GEO,Medical College of Wisconsin,1,0.90312,,0.09896,,0.68885,,0.44175,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42038,SRR5382046,SRX2677137,SRS2075558,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,bmp4 12,GSM2552014,,tissue:whole larval eyes|genotype:Transgenic hsp70l:eGFP bmp4|age:5 dpf,bmp4 12,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:Transgenic hsp70l:eGFP bmp4|age:5 dpf,GSM2552014,GSM2552014: bmp4 12; Danio rerio; RNA Seq,GSM2552014,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552014,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L6_HKZEBxagRAAZRAAPEI-205_1.fq.gz,fastq,796994555.0,16265195.0,GSM2552014 r1,0:49 1:0,A:207611526;C:191999436;G:186148101;T:211228819;N:6673,49,0,,,207611526,191999436,186148101,211228819,6673,SRX2677137,SRS2075558,SRA549807,GEO,Medical College of Wisconsin,1,0.89582,,0.10175,,0.68816,,0.43823,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42039,SRR5382047,SRX2677137,SRS2075558,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,bmp4 12,GSM2552014,,tissue:whole larval eyes|genotype:Transgenic hsp70l:eGFP bmp4|age:5 dpf,bmp4 12,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:Transgenic hsp70l:eGFP bmp4|age:5 dpf,GSM2552014,GSM2552014: bmp4 12; Danio rerio; RNA Seq,GSM2552014,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552014,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L7_HKZEBxagRAAZRAAPEI-205_1.fq.gz,fastq,814617013.0,16624837.0,GSM2552014 r2,0:49 1:0,A:211930527;C:196384410;G:190449227;T:215844442;N:8407,49,0,,,211930527,196384410,190449227,215844442,8407,SRX2677137,SRS2075558,SRA549807,GEO,Medical College of Wisconsin,1,0.9046,,0.1045,,0.68765,,0.44189,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42040,SRR5382044,SRX2677136,SRS2075557,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,bmp4 11,GSM2552013,,tissue:whole larval eyes|genotype:Transgenic hsp70l:eGFP bmp4|age:5 dpf,bmp4 11,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:Transgenic hsp70l:eGFP bmp4|age:5 dpf,GSM2552013,GSM2552013: bmp4 11; Danio rerio; RNA Seq,GSM2552013,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552013,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L6_HKZEBxagRAAXRAAPEI-202_1.fq.gz,fastq,818072248.0,16695352.0,GSM2552013 r1,0:49 1:0,A:213364768;C:196897651;G:191539621;T:216263952;N:6256,49,0,,,213364768,196897651,191539621,216263952,6256,SRX2677136,SRS2075557,SRA549807,GEO,Medical College of Wisconsin,1,0.89563,,0.10696,,0.68828,,0.44425,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42041,SRR5382045,SRX2677136,SRS2075557,SRP102601,PRJNA380770,Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error,GSE97125,Transcriptome Analysis,We compare dissected ocular tissues from wild type and lrp2 /  mutant adult zebrafish in order to examine the genetic pathways underlying the enlarged eye phenotype observed in the absence of Lrp2. ABSTRACT: Mutations in LRP2  a transmembrane receptor  cause ocular enlargement and high myopia. LRP2 is expressed by the RPE and eye ciliary epithelia  binding many extracellular ligands  including Bmp4 and Shh. Signaling mediated by LRP2 is very context dependent  and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled  sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia proper refraction. Using human iPSC derived RPE  and zebrafish  we demonstrate that BACE sheddase dependent LRP2 cleavage produces a soluble domain that binds BMP4  inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand binding receptor forms available: a soluble BMP trap  and a membrane bound RPE signaling facilitator. By modulating LRP2 cleavage  cells can fine tune and coordinate multiple signaling pathways. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth. Overall design: Examination of whole eyes  sclera/choroid  RPE and retina in wild type and lrp2 /  mutant zebrafish at 1 mpf,,,,bmp4 11,GSM2552013,,tissue:whole larval eyes|genotype:Transgenic hsp70l:eGFP bmp4|age:5 dpf,bmp4 11,"Primary sequencing data produced by Illumina HiSeqTM 2000 generates raw reads. Raw reads are subjected to quality control QC to determine if a resequencing step is needed. post QC  raw reads are filtered into clean reads which are aligned to the reference sequences. ""Dirty"" raw reads are reads which contain the sequence of adaptor  high content of unknown bases and low quality reads  and are removed before analysis. Bowtie2  was used to map clean reads to reference gene  and HISAT used to reference genome. The Fragments Per Kilobase of transcript per Million mapped reads FPKM  method is used to calculated expression level. Genome build: GRCz10 GCA 000002035.3 Supplementary files format and content: Excel files containing FKPM values for expression levels.",whole larval eyes,,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,Zebrafish Danio rerio were maintained at 28.5C on an  Aquatic Habitats recirculating filtered water system in reverse osmosis purified water supplemented  with Instant Ocean salts 60 mg/l on a 14 h light: 10 h  dark lighting cycle and fed a standard diet,genotype:Transgenic hsp70l:eGFP bmp4|age:5 dpf,GSM2552013,GSM2552013: bmp4 11; Danio rerio; RNA Seq,GSM2552013,,1,Eyes were dissected were removed  stored overnight at 4C in RNAlater  and RNA was harvested using Trizol reagent. RNA quality was determined using an Agilent BioAnalyzer. RNA libraries were prepared for sequencing using the low input Illumina protocol.,GEO Accession:GSM2552013,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP102601,,,150627_I114_FCH2TT2BBXX_L7_HKZEBxagRAAXRAAPEI-202_1.fq.gz,fastq,901802321.0,18404129.0,GSM2552013 r2,0:49 1:0,A:235005510;C:217168207;G:211265005;T:238355281;N:8318,49,0,,,235005510,217168207,211265005,238355281,8318,SRX2677136,SRS2075557,SRA549807,GEO,Medical College of Wisconsin,1,0.90303,,0.10616,,0.68665,,0.4459,,49,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,bulk,unknown,unknown,,United States,2017-03-28,Larval,Larval,Eye,Sensory System
42930,SRR5833546,SRX3010472,SRS2360156,SRP112616,PRJNA394760,Expression profiling of the retina of pde6c  a zebrafish model of retinal degeneration,GSE101544,Transcriptome Analysis,Retinal degeneration often affects the whole retina even though the disease causing gene is specifically expressed in the light sensitive photoreceptors. These retinal defects can potentially be determined by gene expression profiling of the whole retina. In this study  we measured the gene expression profile of retinas microdissected from a zebrafish pde6cw59 pde6c mutant. Its retinas display not only photoreceptor degeneration but also issues in other cell types starting from 4 dpf dpf. To capture these initial changes  we subjected pde6c and wild type WT retinas at 5 dpf to RNA sequencing RNA Seq on the Illumina HiSeq 2000 platform. The sequencing analyses indicate that the RNA Seq dataset was of high quality. We also validated the RNA Seq results by Reverse Transcription Quantitative Polymerase Chain Reaction RT qPCR of seven phototransduction genes. We found that the fold changes of these genes measured by RT qPCR highly correlated to those measured by RNA Seq. Therefore  our RNA Seq dataset likely captures the molecular changes in the whole pde6c retina. This dataset will facilitate the characterization of the molecular defects in the pde6c retina at the initial stage of retinal degeneration Overall design: 3 samples of pde6c mutant and 3 samples of wild type animals are analyzed.,,pubmed:29231925,,pde6c mutant retina sample 3 PR3,GSM2705949,,source name:retina|tissue:retina|genotype:pde6c mutant|age:5 dpf,pde6c mutant retina sample 3 PR3,Generating genome index using STAR 2.5.3a using the Danio rerio.GRCz10.dna.toplevel.fa and Danio rerio.GRCz10.89.gtf from ensemble ftp Alignment using STAR 2.5.3a Read counting using featureCounts v1.5.2 Generating normalized expression measurements Quantifying differential expression Genome build: Danio rerio.GRCz10.dna.toplevel.fa and Danio rerio.GRCz10.89.gtf from ensemble ftp For more analysis done  see https://gist.github.com/coralzhang/fc4e51609ff316486c1682feed6404a9/471afb15b7f7b230a38e4eedaadcf5f679412a07 Supplementary files format and content: text file with raw read counts or FPKM,retina,,Total RNAs was extracted from the biological replicates by an optimized procedure that combined TRIzol Life Technologies  Grand Island  NY and RNeasy Micro kit Qiagen  Valencia  CA. The quality of the extracted total RNAs were evaluated by Bioanalyzer electrophoresis Agilent Technologies  Santa Clara  CA. Libraries were prepared according to Illumina's instructions.,,tissue:retina|genotype:pde6c mutant|age:5 dpf,GSM2705949,GSM2705949: pde6c mutant retina sample 3 PR3; Danio rerio; RNA Seq,GSM2705949,,1,Total RNAs was extracted from the biological replicates by an optimized procedure that combined TRIzol Life Technologies  Grand Island  NY and RNeasy Micro kit Qiagen  Valencia  CA. The quality of the extracted total RNAs were evaluated by Bioanalyzer electrophoresis Agilent Technologies  Santa Clara  CA. Libraries were prepared according to Illumina's instructions.,GEO Accession:GSM2705949,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,SRP112616,,,trim-PR3-R1.fastq.gz trim-PR3-R2.fastq.gz,fastq fastq,9267632803.0,47008978.0,GSM2705949 r1,0:98.63 1:98.51,A:2750256605;C:1769304271;G:1860827242;T:2887067967;N:176718,98,98,,,2750256605,1769304271,1860827242,2887067967,176718,SRX3010472,SRS2360156,SRA587806,GEO,"Department of Statistics, University of Georgia",2,0.79966,0.78774,0.47769,0.47298,0.76487,0.76712,0.50124,0.52896,101,101,B,B,biological fallback assumption,illumina,hiseq_era,unknown,random_priming,unknown,bulk,unknown,unknown,,United States,2017-07-17,Larval,Larval,Eye,Sensory System
42931,SRR5833545,SRX3010471,SRS2360155,SRP112616,PRJNA394760,Expression profiling of the retina of pde6c  a zebrafish model of retinal degeneration,GSE101544,Transcriptome Analysis,Retinal degeneration often affects the whole retina even though the disease causing gene is specifically expressed in the light sensitive photoreceptors. These retinal defects can potentially be determined by gene expression profiling of the whole retina. In this study  we measured the gene expression profile of retinas microdissected from a zebrafish pde6cw59 pde6c mutant. Its retinas display not only photoreceptor degeneration but also issues in other cell types starting from 4 dpf dpf. To capture these initial changes  we subjected pde6c and wild type WT retinas at 5 dpf to RNA sequencing RNA Seq on the Illumina HiSeq 2000 platform. The sequencing analyses indicate that the RNA Seq dataset was of high quality. We also validated the RNA Seq results by Reverse Transcription Quantitative Polymerase Chain Reaction RT qPCR of seven phototransduction genes. We found that the fold changes of these genes measured by RT qPCR highly correlated to those measured by RNA Seq. Therefore  our RNA Seq dataset likely captures the molecular changes in the whole pde6c retina. This dataset will facilitate the characterization of the molecular defects in the pde6c retina at the initial stage of retinal degeneration Overall design: 3 samples of pde6c mutant and 3 samples of wild type animals are analyzed.,,pubmed:29231925,,pde6c mutant retina sample 2 PR2,GSM2705948,,source name:retina|tissue:retina|genotype:pde6c mutant|age:5 dpf,pde6c mutant retina sample 2 PR2,Generating genome index using STAR 2.5.3a using the Danio rerio.GRCz10.dna.toplevel.fa and Danio rerio.GRCz10.89.gtf from ensemble ftp Alignment using STAR 2.5.3a Read counting using featureCounts v1.5.2 Generating normalized expression measurements Quantifying differential expression Genome build: Danio rerio.GRCz10.dna.toplevel.fa and Danio rerio.GRCz10.89.gtf from ensemble ftp For more analysis done  see https://gist.github.com/coralzhang/fc4e51609ff316486c1682feed6404a9/471afb15b7f7b230a38e4eedaadcf5f679412a07 Supplementary files format and content: text file with raw read counts or FPKM,retina,,Total RNAs was extracted from the biological replicates by an optimized procedure that combined TRIzol Life Technologies  Grand Island  NY and RNeasy Micro kit Qiagen  Valencia  CA. The quality of the extracted total RNAs were evaluated by Bioanalyzer electrophoresis Agilent Technologies  Santa Clara  CA. Libraries were prepared according to Illumina's instructions.,,tissue:retina|genotype:pde6c mutant|age:5 dpf,GSM2705948,GSM2705948: pde6c mutant retina sample 2 PR2; Danio rerio; RNA Seq,GSM2705948,,1,Total RNAs was extracted from the biological replicates by an optimized procedure that combined TRIzol Life Technologies  Grand Island  NY and RNeasy Micro kit Qiagen  Valencia  CA. The quality of the extracted total RNAs were evaluated by Bioanalyzer electrophoresis Agilent Technologies  Santa Clara  CA. Libraries were prepared according to Illumina's instructions.,GEO Accession:GSM2705948,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,SRP112616,,,trim-PR2-R1.fastq.gz trim-PR2-R2.fastq.gz,fastq fastq,9200373440.0,46550508.0,GSM2705948 r1,0:98.75 1:98.89,A:2724941180;C:1777010271;G:1859753250;T:2838492418;N:176321,98,98,,,2724941180,1777010271,1859753250,2838492418,176321,SRX3010471,SRS2360155,SRA587806,GEO,"Department of Statistics, University of Georgia",2,0.80793,0.797,0.47529,0.47189,0.7628,0.76439,0.53576,0.53345,101,101,B,B,biological fallback assumption,illumina,hiseq_era,unknown,random_priming,unknown,bulk,unknown,unknown,,United States,2017-07-17,Larval,Larval,Eye,Sensory System
42932,SRR5833544,SRX3010470,SRS2360153,SRP112616,PRJNA394760,Expression profiling of the retina of pde6c  a zebrafish model of retinal degeneration,GSE101544,Transcriptome Analysis,Retinal degeneration often affects the whole retina even though the disease causing gene is specifically expressed in the light sensitive photoreceptors. These retinal defects can potentially be determined by gene expression profiling of the whole retina. In this study  we measured the gene expression profile of retinas microdissected from a zebrafish pde6cw59 pde6c mutant. Its retinas display not only photoreceptor degeneration but also issues in other cell types starting from 4 dpf dpf. To capture these initial changes  we subjected pde6c and wild type WT retinas at 5 dpf to RNA sequencing RNA Seq on the Illumina HiSeq 2000 platform. The sequencing analyses indicate that the RNA Seq dataset was of high quality. We also validated the RNA Seq results by Reverse Transcription Quantitative Polymerase Chain Reaction RT qPCR of seven phototransduction genes. We found that the fold changes of these genes measured by RT qPCR highly correlated to those measured by RNA Seq. Therefore  our RNA Seq dataset likely captures the molecular changes in the whole pde6c retina. This dataset will facilitate the characterization of the molecular defects in the pde6c retina at the initial stage of retinal degeneration Overall design: 3 samples of pde6c mutant and 3 samples of wild type animals are analyzed.,,pubmed:29231925,,pde6c mutant retina sample 1 PR1,GSM2705947,,source name:retina|tissue:retina|genotype:pde6c mutant|age:5 dpf,pde6c mutant retina sample 1 PR1,Generating genome index using STAR 2.5.3a using the Danio rerio.GRCz10.dna.toplevel.fa and Danio rerio.GRCz10.89.gtf from ensemble ftp Alignment using STAR 2.5.3a Read counting using featureCounts v1.5.2 Generating normalized expression measurements Quantifying differential expression Genome build: Danio rerio.GRCz10.dna.toplevel.fa and Danio rerio.GRCz10.89.gtf from ensemble ftp For more analysis done  see https://gist.github.com/coralzhang/fc4e51609ff316486c1682feed6404a9/471afb15b7f7b230a38e4eedaadcf5f679412a07 Supplementary files format and content: text file with raw read counts or FPKM,retina,,Total RNAs was extracted from the biological replicates by an optimized procedure that combined TRIzol Life Technologies  Grand Island  NY and RNeasy Micro kit Qiagen  Valencia  CA. The quality of the extracted total RNAs were evaluated by Bioanalyzer electrophoresis Agilent Technologies  Santa Clara  CA. Libraries were prepared according to Illumina's instructions.,,tissue:retina|genotype:pde6c mutant|age:5 dpf,GSM2705947,GSM2705947: pde6c mutant retina sample 1 PR1; Danio rerio; RNA Seq,GSM2705947,,1,Total RNAs was extracted from the biological replicates by an optimized procedure that combined TRIzol Life Technologies  Grand Island  NY and RNeasy Micro kit Qiagen  Valencia  CA. The quality of the extracted total RNAs were evaluated by Bioanalyzer electrophoresis Agilent Technologies  Santa Clara  CA. Libraries were prepared according to Illumina's instructions.,GEO Accession:GSM2705947,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,SRP112616,,,trim-PR1-R1.fastq.gz trim-PR1-R2.fastq.gz,fastq fastq,9677531137.0,48960995.0,GSM2705947 r1,0:98.85 1:98.81,A:2823942389;C:1921043215;G:1995590627;T:2936770057;N:184849,98,98,,,2823942389,1921043215,1995590627,2936770057,184849,SRX3010470,SRS2360153,SRA587806,GEO,"Department of Statistics, University of Georgia",2,0.81627,0.80533,0.43663,0.43372,0.75487,0.75574,0.55341,0.55312,101,101,B,B,biological fallback assumption,illumina,hiseq_era,unknown,random_priming,unknown,bulk,unknown,unknown,,United States,2017-07-17,Larval,Larval,Eye,Sensory System
42933,SRR5833543,SRX3010469,SRS2360154,SRP112616,PRJNA394760,Expression profiling of the retina of pde6c  a zebrafish model of retinal degeneration,GSE101544,Transcriptome Analysis,Retinal degeneration often affects the whole retina even though the disease causing gene is specifically expressed in the light sensitive photoreceptors. These retinal defects can potentially be determined by gene expression profiling of the whole retina. In this study  we measured the gene expression profile of retinas microdissected from a zebrafish pde6cw59 pde6c mutant. Its retinas display not only photoreceptor degeneration but also issues in other cell types starting from 4 dpf dpf. To capture these initial changes  we subjected pde6c and wild type WT retinas at 5 dpf to RNA sequencing RNA Seq on the Illumina HiSeq 2000 platform. The sequencing analyses indicate that the RNA Seq dataset was of high quality. We also validated the RNA Seq results by Reverse Transcription Quantitative Polymerase Chain Reaction RT qPCR of seven phototransduction genes. We found that the fold changes of these genes measured by RT qPCR highly correlated to those measured by RNA Seq. Therefore  our RNA Seq dataset likely captures the molecular changes in the whole pde6c retina. This dataset will facilitate the characterization of the molecular defects in the pde6c retina at the initial stage of retinal degeneration Overall design: 3 samples of pde6c mutant and 3 samples of wild type animals are analyzed.,,pubmed:29231925,,wild type retina sample 3 WR3,GSM2705946,,source name:retina|tissue:retina|genotype:wildtype|age:5 dpf,wild type retina sample 3 WR3,Generating genome index using STAR 2.5.3a using the Danio rerio.GRCz10.dna.toplevel.fa and Danio rerio.GRCz10.89.gtf from ensemble ftp Alignment using STAR 2.5.3a Read counting using featureCounts v1.5.2 Generating normalized expression measurements Quantifying differential expression Genome build: Danio rerio.GRCz10.dna.toplevel.fa and Danio rerio.GRCz10.89.gtf from ensemble ftp For more analysis done  see https://gist.github.com/coralzhang/fc4e51609ff316486c1682feed6404a9/471afb15b7f7b230a38e4eedaadcf5f679412a07 Supplementary files format and content: text file with raw read counts or FPKM,retina,,Total RNAs was extracted from the biological replicates by an optimized procedure that combined TRIzol Life Technologies  Grand Island  NY and RNeasy Micro kit Qiagen  Valencia  CA. The quality of the extracted total RNAs were evaluated by Bioanalyzer electrophoresis Agilent Technologies  Santa Clara  CA. Libraries were prepared according to Illumina's instructions.,,tissue:retina|genotype:wildtype|age:5 dpf,GSM2705946,GSM2705946: wild type retina sample 3 WR3; Danio rerio; RNA Seq,GSM2705946,,1,Total RNAs was extracted from the biological replicates by an optimized procedure that combined TRIzol Life Technologies  Grand Island  NY and RNeasy Micro kit Qiagen  Valencia  CA. The quality of the extracted total RNAs were evaluated by Bioanalyzer electrophoresis Agilent Technologies  Santa Clara  CA. Libraries were prepared according to Illumina's instructions.,GEO Accession:GSM2705946,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,SRP112616,,,trim-WR3-R2.fastq.gz trim-WR3-R1.fastq.gz,fastq fastq,9774473496.0,49621925.0,GSM2705946 r1,0:98.62 1:98.36,A:2910744713;C:1863135495;G:1956081537;T:3044325954;N:185797,98,98,,,2910744713,1863135495,1956081537,3044325954,185797,SRX3010469,SRS2360154,SRA587806,GEO,"Department of Statistics, University of Georgia",2,0.79934,0.78841,0.47972,0.476,0.76524,0.76978,0.52583,0.52942,101,101,B,B,biological fallback assumption,illumina,hiseq_era,unknown,random_priming,unknown,bulk,unknown,unknown,,United States,2017-07-17,Larval,Larval,Eye,Sensory System
42934,SRR5833542,SRX3010468,SRS2360151,SRP112616,PRJNA394760,Expression profiling of the retina of pde6c  a zebrafish model of retinal degeneration,GSE101544,Transcriptome Analysis,Retinal degeneration often affects the whole retina even though the disease causing gene is specifically expressed in the light sensitive photoreceptors. These retinal defects can potentially be determined by gene expression profiling of the whole retina. In this study  we measured the gene expression profile of retinas microdissected from a zebrafish pde6cw59 pde6c mutant. Its retinas display not only photoreceptor degeneration but also issues in other cell types starting from 4 dpf dpf. To capture these initial changes  we subjected pde6c and wild type WT retinas at 5 dpf to RNA sequencing RNA Seq on the Illumina HiSeq 2000 platform. The sequencing analyses indicate that the RNA Seq dataset was of high quality. We also validated the RNA Seq results by Reverse Transcription Quantitative Polymerase Chain Reaction RT qPCR of seven phototransduction genes. We found that the fold changes of these genes measured by RT qPCR highly correlated to those measured by RNA Seq. Therefore  our RNA Seq dataset likely captures the molecular changes in the whole pde6c retina. This dataset will facilitate the characterization of the molecular defects in the pde6c retina at the initial stage of retinal degeneration Overall design: 3 samples of pde6c mutant and 3 samples of wild type animals are analyzed.,,pubmed:29231925,,wild type retina sample 2 WR2,GSM2705945,,source name:retina|tissue:retina|genotype:wildtype|age:5 dpf,wild type retina sample 2 WR2,Generating genome index using STAR 2.5.3a using the Danio rerio.GRCz10.dna.toplevel.fa and Danio rerio.GRCz10.89.gtf from ensemble ftp Alignment using STAR 2.5.3a Read counting using featureCounts v1.5.2 Generating normalized expression measurements Quantifying differential expression Genome build: Danio rerio.GRCz10.dna.toplevel.fa and Danio rerio.GRCz10.89.gtf from ensemble ftp For more analysis done  see https://gist.github.com/coralzhang/fc4e51609ff316486c1682feed6404a9/471afb15b7f7b230a38e4eedaadcf5f679412a07 Supplementary files format and content: text file with raw read counts or FPKM,retina,,Total RNAs was extracted from the biological replicates by an optimized procedure that combined TRIzol Life Technologies  Grand Island  NY and RNeasy Micro kit Qiagen  Valencia  CA. The quality of the extracted total RNAs were evaluated by Bioanalyzer electrophoresis Agilent Technologies  Santa Clara  CA. Libraries were prepared according to Illumina's instructions.,,tissue:retina|genotype:wildtype|age:5 dpf,GSM2705945,GSM2705945: wild type retina sample 2 WR2; Danio rerio; RNA Seq,GSM2705945,,1,Total RNAs was extracted from the biological replicates by an optimized procedure that combined TRIzol Life Technologies  Grand Island  NY and RNeasy Micro kit Qiagen  Valencia  CA. The quality of the extracted total RNAs were evaluated by Bioanalyzer electrophoresis Agilent Technologies  Santa Clara  CA. Libraries were prepared according to Illumina's instructions.,GEO Accession:GSM2705945,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,SRP112616,,,trim-WR2-R1.fastq.gz trim-WR2-R2.fastq.gz,fastq fastq,8477336082.0,42986393.0,GSM2705945 r1,0:98.65 1:98.56,A:2512079309;C:1596476120;G:1697011877;T:2671608220;N:160556,98,98,,,2512079309,1596476120,1697011877,2671608220,160556,SRX3010468,SRS2360151,SRA587806,GEO,"Department of Statistics, University of Georgia",2,0.79778,0.78411,0.47086,0.46672,0.77043,0.77339,0.51627,0.52136,101,64,B,B,biological fallback assumption,illumina,hiseq_era,unknown,random_priming,unknown,bulk,unknown,unknown,,United States,2017-07-17,Larval,Larval,Eye,Sensory System
42935,SRR5833541,SRX3010467,SRS2360152,SRP112616,PRJNA394760,Expression profiling of the retina of pde6c  a zebrafish model of retinal degeneration,GSE101544,Transcriptome Analysis,Retinal degeneration often affects the whole retina even though the disease causing gene is specifically expressed in the light sensitive photoreceptors. These retinal defects can potentially be determined by gene expression profiling of the whole retina. In this study  we measured the gene expression profile of retinas microdissected from a zebrafish pde6cw59 pde6c mutant. Its retinas display not only photoreceptor degeneration but also issues in other cell types starting from 4 dpf dpf. To capture these initial changes  we subjected pde6c and wild type WT retinas at 5 dpf to RNA sequencing RNA Seq on the Illumina HiSeq 2000 platform. The sequencing analyses indicate that the RNA Seq dataset was of high quality. We also validated the RNA Seq results by Reverse Transcription Quantitative Polymerase Chain Reaction RT qPCR of seven phototransduction genes. We found that the fold changes of these genes measured by RT qPCR highly correlated to those measured by RNA Seq. Therefore  our RNA Seq dataset likely captures the molecular changes in the whole pde6c retina. This dataset will facilitate the characterization of the molecular defects in the pde6c retina at the initial stage of retinal degeneration Overall design: 3 samples of pde6c mutant and 3 samples of wild type animals are analyzed.,,pubmed:29231925,,wild type retina sample 1 WR1,GSM2705944,,source name:retina|tissue:retina|genotype:wildtype|age:5 dpf,wild type retina sample 1 WR1,Generating genome index using STAR 2.5.3a using the Danio rerio.GRCz10.dna.toplevel.fa and Danio rerio.GRCz10.89.gtf from ensemble ftp Alignment using STAR 2.5.3a Read counting using featureCounts v1.5.2 Generating normalized expression measurements with cufflinks 2.2.1 Quantifying differential expression Genome build: Danio rerio.GRCz10.dna.toplevel.fa and Danio rerio.GRCz10.89.gtf from ensemble ftp For more analysis done  see https://gist.github.com/coralzhang/fc4e51609ff316486c1682feed6404a9/471afb15b7f7b230a38e4eedaadcf5f679412a07 Supplementary files format and content: text file with raw read counts or FPKM,retina,,Total RNAs was extracted from the biological replicates by an optimized procedure that combined TRIzol Life Technologies  Grand Island  NY and RNeasy Micro kit Qiagen  Valencia  CA. The quality of the extracted total RNAs were evaluated by Bioanalyzer electrophoresis Agilent Technologies  Santa Clara  CA. Libraries were prepared according to Illumina's instructions.,,tissue:retina|genotype:wildtype|age:5 dpf,GSM2705944,GSM2705944: wild type retina sample 1 WR1; Danio rerio; RNA Seq,GSM2705944,,1,Total RNAs was extracted from the biological replicates by an optimized procedure that combined TRIzol Life Technologies  Grand Island  NY and RNeasy Micro kit Qiagen  Valencia  CA. The quality of the extracted total RNAs were evaluated by Bioanalyzer electrophoresis Agilent Technologies  Santa Clara  CA. Libraries were prepared according to Illumina's instructions.,GEO Accession:GSM2705944,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2000,,SRP112616,,,trim-WR1-R2.fastq.gz trim-WR1-R1.fastq.gz,fastq fastq,10078850313.0,51108456.0,GSM2705944 r1,0:98.66 1:98.55,A:2896366580;C:2023834885;G:2117794332;T:3040663699;N:190817,98,98,,,2896366580,2023834885,2117794332,3040663699,190817,SRX3010467,SRS2360152,SRA587806,GEO,"Department of Statistics, University of Georgia",2,0.81956,0.80495,0.41661,0.41171,0.75773,0.7599,0.54429,0.54605,101,101,B,B,biological fallback assumption,illumina,hiseq_era,unknown,random_priming,unknown,bulk,unknown,unknown,,United States,2017-07-17,Larval,Larval,Eye,Sensory System
49296,SRR7886639,SRX4724291,SRS3809064,SRP162288,PRJNA492363,Genetic control of cellular morphogenesis in Müller glia,GSE120275,Transcriptome Analysis,How the various cell types of the body achieve their specific shapes is fundamentally unknown.  Here  we explore this issue by identifying genes involved in the elaboration of the complex  yet conserved  cellular morphology of Müller glial MG cells in the retina.  Using genomic  based strategies in zebrafish  we found more than 40 candidate genes involved in specific aspects of MG morphogenesis.  The successive steps of cell morphogenesis correlate with the timing of the expression of cohorts of inter related genes that have roles in generating the particular anatomical features of these cells  suggesting that a sequence of genetic regulomes govern stepwise cellular morphogenesis in this system. Overall design: 12 samples with three replicates each are provided.  GFAP:GFP positive and negative cells were FAC sorted from wild type animals from each developmental stage,,pubmed:30924555,,192MGS3,GSM3397473,,source name:192hpf GFAP:GFP Positive|age:192hpf|genotype:Control GFAP:GFP|tissue:Dissociated retinal tissue|gfap:Positive,192MGS3,Trimmomatic read trimming Hisat2 genome aligment Samtools Sam to Bam sorting Bioconuducter Feature counts  Rsubread  limma DESeq2  DEFormats  org.Dr.eg.db  EdgeR Genome build: GRCz10 Supplementary files format and content: TMM fold change analysis,192hpf GFAP:GFP Positive,,Single cell suspensions were sorted on a Beckman Coulter MoFlo to capture Muller glia GFP and control retinal tissue non GFP. Cells were sorted into lysis buffer  and RNA was immediately extracted using the RNeasy mini kit Qiagen. RNA concentration and qualities were assessed on an Agilent Bioanalyzer and RNA amplification and cDNA synthesis was performed with the Ovation RNA Amplification System V2 NuGEN using manufacturer’s protocol. Nextera library preparations were performed using the Nextera DNA library kit according to the manufacturer's directions,,age:192hpf|genotype:Control GFAP:GFP|tissue:Dissociated retinal tissue|gfap:Positive,GSM3397473,GSM3397473: 192MGS3; Danio rerio; RNA Seq,GSM3397473,,1,Single cell suspensions were sorted on a Beckman Coulter MoFlo to capture Muller glia GFP and control retinal tissue non GFP. Cells were sorted into lysis buffer  and RNA was immediately extracted using the RNeasy mini kit Qiagen. RNA concentration and qualities were assessed on an Agilent Bioanalyzer and RNA amplification and cDNA synthesis was performed with the Ovation RNA Amplification System V2 NuGEN using manufacturer's protocol. Nextera library preparations were performed using the Nextera DNA library kit according to the manufacturer's directions,GEO Accession:GSM3397473,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2500,,SRP162288,,dangling references:treat as unmapped,192MGS3.bam,bam,2998192500.0,23985540.0,GSM3397473 r1,0:125,A:785328976;C:735689839;G:683280598;T:793875354;N:17733,125,,,,785328976,735689839,683280598,793875354,17733,SRX4724291,SRS3809064,SRA780762,GEO,University of Cambridge,1,0.37806,,0.04299,,0.83522,,0.61667,,125,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,nextera,bulk,unknown,unknown,,United Kingdom,2018-09-20,Larval,Larval,Eye,Sensory System
49297,SRR7886638,SRX4724290,SRS3809063,SRP162288,PRJNA492363,Genetic control of cellular morphogenesis in Müller glia,GSE120275,Transcriptome Analysis,How the various cell types of the body achieve their specific shapes is fundamentally unknown.  Here  we explore this issue by identifying genes involved in the elaboration of the complex  yet conserved  cellular morphology of Müller glial MG cells in the retina.  Using genomic  based strategies in zebrafish  we found more than 40 candidate genes involved in specific aspects of MG morphogenesis.  The successive steps of cell morphogenesis correlate with the timing of the expression of cohorts of inter related genes that have roles in generating the particular anatomical features of these cells  suggesting that a sequence of genetic regulomes govern stepwise cellular morphogenesis in this system. Overall design: 12 samples with three replicates each are provided.  GFAP:GFP positive and negative cells were FAC sorted from wild type animals from each developmental stage,,pubmed:30924555,,192MGS2,GSM3397472,,source name:192hpf GFAP:GFP Positive|age:192hpf|genotype:Control GFAP:GFP|tissue:Dissociated retinal tissue|gfap:Positive,192MGS2,Trimmomatic read trimming Hisat2 genome aligment Samtools Sam to Bam sorting Bioconuducter Feature counts  Rsubread  limma DESeq2  DEFormats  org.Dr.eg.db  EdgeR Genome build: GRCz10 Supplementary files format and content: TMM fold change analysis,192hpf GFAP:GFP Positive,,Single cell suspensions were sorted on a Beckman Coulter MoFlo to capture Muller glia GFP and control retinal tissue non GFP. Cells were sorted into lysis buffer  and RNA was immediately extracted using the RNeasy mini kit Qiagen. RNA concentration and qualities were assessed on an Agilent Bioanalyzer and RNA amplification and cDNA synthesis was performed with the Ovation RNA Amplification System V2 NuGEN using manufacturer’s protocol. Nextera library preparations were performed using the Nextera DNA library kit according to the manufacturer's directions,,age:192hpf|genotype:Control GFAP:GFP|tissue:Dissociated retinal tissue|gfap:Positive,GSM3397472,GSM3397472: 192MGS2; Danio rerio; RNA Seq,GSM3397472,,1,Single cell suspensions were sorted on a Beckman Coulter MoFlo to capture Muller glia GFP and control retinal tissue non GFP. Cells were sorted into lysis buffer  and RNA was immediately extracted using the RNeasy mini kit Qiagen. RNA concentration and qualities were assessed on an Agilent Bioanalyzer and RNA amplification and cDNA synthesis was performed with the Ovation RNA Amplification System V2 NuGEN using manufacturer's protocol. Nextera library preparations were performed using the Nextera DNA library kit according to the manufacturer's directions,GEO Accession:GSM3397472,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2500,,SRP162288,,dangling references:treat as unmapped,192MGS2.bam,bam,2964338500.0,23714708.0,GSM3397472 r1,0:125,A:771195405;C:723851427;G:682149646;T:787129917;N:12105,125,,,,771195405,723851427,682149646,787129917,12105,SRX4724290,SRS3809063,SRA780762,GEO,University of Cambridge,1,0.36196,,0.04304,,0.84682,,0.62523,,125,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,nextera,bulk,unknown,unknown,,United Kingdom,2018-09-20,Larval,Larval,Eye,Sensory System
49298,SRR7886637,SRX4724289,SRS3809065,SRP162288,PRJNA492363,Genetic control of cellular morphogenesis in Müller glia,GSE120275,Transcriptome Analysis,How the various cell types of the body achieve their specific shapes is fundamentally unknown.  Here  we explore this issue by identifying genes involved in the elaboration of the complex  yet conserved  cellular morphology of Müller glial MG cells in the retina.  Using genomic  based strategies in zebrafish  we found more than 40 candidate genes involved in specific aspects of MG morphogenesis.  The successive steps of cell morphogenesis correlate with the timing of the expression of cohorts of inter related genes that have roles in generating the particular anatomical features of these cells  suggesting that a sequence of genetic regulomes govern stepwise cellular morphogenesis in this system. Overall design: 12 samples with three replicates each are provided.  GFAP:GFP positive and negative cells were FAC sorted from wild type animals from each developmental stage,,pubmed:30924555,,192MGS1,GSM3397471,,source name:192hpf GFAP:GFP Positive|age:192hpf|genotype:Control GFAP:GFP|tissue:Dissociated retinal tissue|gfap:Positive,192MGS1,Trimmomatic read trimming Hisat2 genome aligment Samtools Sam to Bam sorting Bioconuducter Feature counts  Rsubread  limma DESeq2  DEFormats  org.Dr.eg.db  EdgeR Genome build: GRCz10 Supplementary files format and content: TMM fold change analysis,192hpf GFAP:GFP Positive,,Single cell suspensions were sorted on a Beckman Coulter MoFlo to capture Muller glia GFP and control retinal tissue non GFP. Cells were sorted into lysis buffer  and RNA was immediately extracted using the RNeasy mini kit Qiagen. RNA concentration and qualities were assessed on an Agilent Bioanalyzer and RNA amplification and cDNA synthesis was performed with the Ovation RNA Amplification System V2 NuGEN using manufacturer’s protocol. Nextera library preparations were performed using the Nextera DNA library kit according to the manufacturer's directions,,age:192hpf|genotype:Control GFAP:GFP|tissue:Dissociated retinal tissue|gfap:Positive,GSM3397471,GSM3397471: 192MGS1; Danio rerio; RNA Seq,GSM3397471,,1,Single cell suspensions were sorted on a Beckman Coulter MoFlo to capture Muller glia GFP and control retinal tissue non GFP. Cells were sorted into lysis buffer  and RNA was immediately extracted using the RNeasy mini kit Qiagen. RNA concentration and qualities were assessed on an Agilent Bioanalyzer and RNA amplification and cDNA synthesis was performed with the Ovation RNA Amplification System V2 NuGEN using manufacturer's protocol. Nextera library preparations were performed using the Nextera DNA library kit according to the manufacturer's directions,GEO Accession:GSM3397471,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2500,,SRP162288,,dangling references:treat as unmapped,192MGS1.bam,bam,2964338500.0,23714708.0,GSM3397471 r1,0:125,A:776632663;C:727429819;G:673884321;T:786325865;N:65832,125,,,,776632663,727429819,673884321,786325865,65832,SRX4724289,SRS3809065,SRA780762,GEO,University of Cambridge,1,0.36617,,0.04341,,0.8406,,0.6199,,125,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,nextera,bulk,unknown,unknown,,United Kingdom,2018-09-20,Larval,Larval,Eye,Sensory System
49299,SRR7886636,SRX4724288,SRS3809062,SRP162288,PRJNA492363,Genetic control of cellular morphogenesis in Müller glia,GSE120275,Transcriptome Analysis,How the various cell types of the body achieve their specific shapes is fundamentally unknown.  Here  we explore this issue by identifying genes involved in the elaboration of the complex  yet conserved  cellular morphology of Müller glial MG cells in the retina.  Using genomic  based strategies in zebrafish  we found more than 40 candidate genes involved in specific aspects of MG morphogenesis.  The successive steps of cell morphogenesis correlate with the timing of the expression of cohorts of inter related genes that have roles in generating the particular anatomical features of these cells  suggesting that a sequence of genetic regulomes govern stepwise cellular morphogenesis in this system. Overall design: 12 samples with three replicates each are provided.  GFAP:GFP positive and negative cells were FAC sorted from wild type animals from each developmental stage,,pubmed:30924555,,192CS3,GSM3397470,,source name:192hpf GFAP:GFP Negative|age:192hpf|genotype:Control GFAP:GFP|tissue:Dissociated retinal tissue|gfap:Negative,192CS3,Trimmomatic read trimming Hisat2 genome aligment Samtools Sam to Bam sorting Bioconuducter Feature counts  Rsubread  limma DESeq2  DEFormats  org.Dr.eg.db  EdgeR Genome build: GRCz10 Supplementary files format and content: TMM fold change analysis,192hpf GFAP:GFP Negative,,Single cell suspensions were sorted on a Beckman Coulter MoFlo to capture Muller glia GFP and control retinal tissue non GFP. Cells were sorted into lysis buffer  and RNA was immediately extracted using the RNeasy mini kit Qiagen. RNA concentration and qualities were assessed on an Agilent Bioanalyzer and RNA amplification and cDNA synthesis was performed with the Ovation RNA Amplification System V2 NuGEN using manufacturer’s protocol. Nextera library preparations were performed using the Nextera DNA library kit according to the manufacturer's directions,,age:192hpf|genotype:Control GFAP:GFP|tissue:Dissociated retinal tissue|gfap:Negative,GSM3397470,GSM3397470: 192CS3; Danio rerio; RNA Seq,GSM3397470,,1,Single cell suspensions were sorted on a Beckman Coulter MoFlo to capture Muller glia GFP and control retinal tissue non GFP. Cells were sorted into lysis buffer  and RNA was immediately extracted using the RNeasy mini kit Qiagen. RNA concentration and qualities were assessed on an Agilent Bioanalyzer and RNA amplification and cDNA synthesis was performed with the Ovation RNA Amplification System V2 NuGEN using manufacturer's protocol. Nextera library preparations were performed using the Nextera DNA library kit according to the manufacturer's directions,GEO Accession:GSM3397470,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2500,,SRP162288,,dangling references:treat as unmapped,192CS3.bam,bam,3253316125.0,26026529.0,GSM3397470 r1,0:125,A:843188450;C:804209262;G:721349992;T:884549914;N:18507,125,,,,843188450,804209262,721349992,884549914,18507,SRX4724288,SRS3809062,SRA780762,GEO,University of Cambridge,1,0.41789,,0.05889,,0.7933,,0.56031,,125,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,nextera,bulk,unknown,unknown,,United Kingdom,2018-09-20,Larval,Larval,Eye,Sensory System
49300,SRR7886635,SRX4724287,SRS3809060,SRP162288,PRJNA492363,Genetic control of cellular morphogenesis in Müller glia,GSE120275,Transcriptome Analysis,How the various cell types of the body achieve their specific shapes is fundamentally unknown.  Here  we explore this issue by identifying genes involved in the elaboration of the complex  yet conserved  cellular morphology of Müller glial MG cells in the retina.  Using genomic  based strategies in zebrafish  we found more than 40 candidate genes involved in specific aspects of MG morphogenesis.  The successive steps of cell morphogenesis correlate with the timing of the expression of cohorts of inter related genes that have roles in generating the particular anatomical features of these cells  suggesting that a sequence of genetic regulomes govern stepwise cellular morphogenesis in this system. Overall design: 12 samples with three replicates each are provided.  GFAP:GFP positive and negative cells were FAC sorted from wild type animals from each developmental stage,,pubmed:30924555,,192CS2,GSM3397469,,source name:192hpf GFAP:GFP Negative|age:192hpf|genotype:Control GFAP:GFP|tissue:Dissociated retinal tissue|gfap:Negative,192CS2,Trimmomatic read trimming Hisat2 genome aligment Samtools Sam to Bam sorting Bioconuducter Feature counts  Rsubread  limma DESeq2  DEFormats  org.Dr.eg.db  EdgeR Genome build: GRCz10 Supplementary files format and content: TMM fold change analysis,192hpf GFAP:GFP Negative,,Single cell suspensions were sorted on a Beckman Coulter MoFlo to capture Muller glia GFP and control retinal tissue non GFP. Cells were sorted into lysis buffer  and RNA was immediately extracted using the RNeasy mini kit Qiagen. RNA concentration and qualities were assessed on an Agilent Bioanalyzer and RNA amplification and cDNA synthesis was performed with the Ovation RNA Amplification System V2 NuGEN using manufacturer’s protocol. Nextera library preparations were performed using the Nextera DNA library kit according to the manufacturer's directions,,age:192hpf|genotype:Control GFAP:GFP|tissue:Dissociated retinal tissue|gfap:Negative,GSM3397469,GSM3397469: 192CS2; Danio rerio; RNA Seq,GSM3397469,,1,Single cell suspensions were sorted on a Beckman Coulter MoFlo to capture Muller glia GFP and control retinal tissue non GFP. Cells were sorted into lysis buffer  and RNA was immediately extracted using the RNeasy mini kit Qiagen. RNA concentration and qualities were assessed on an Agilent Bioanalyzer and RNA amplification and cDNA synthesis was performed with the Ovation RNA Amplification System V2 NuGEN using manufacturer's protocol. Nextera library preparations were performed using the Nextera DNA library kit according to the manufacturer's directions,GEO Accession:GSM3397469,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2500,,SRP162288,,dangling references:treat as unmapped,192CS2.bam,bam,3097074000.0,24776592.0,GSM3397469 r1,0:125,A:816743917;C:748031875;G:702166584;T:830119487;N:12137,125,,,,816743917,748031875,702166584,830119487,12137,SRX4724287,SRS3809060,SRA780762,GEO,University of Cambridge,1,0.41038,,0.05892,,0.80353,,0.56676,,125,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,nextera,bulk,unknown,unknown,,United Kingdom,2018-09-20,Larval,Larval,Eye,Sensory System
49301,SRR7886634,SRX4724286,SRS3809061,SRP162288,PRJNA492363,Genetic control of cellular morphogenesis in Müller glia,GSE120275,Transcriptome Analysis,How the various cell types of the body achieve their specific shapes is fundamentally unknown.  Here  we explore this issue by identifying genes involved in the elaboration of the complex  yet conserved  cellular morphology of Müller glial MG cells in the retina.  Using genomic  based strategies in zebrafish  we found more than 40 candidate genes involved in specific aspects of MG morphogenesis.  The successive steps of cell morphogenesis correlate with the timing of the expression of cohorts of inter related genes that have roles in generating the particular anatomical features of these cells  suggesting that a sequence of genetic regulomes govern stepwise cellular morphogenesis in this system. Overall design: 12 samples with three replicates each are provided.  GFAP:GFP positive and negative cells were FAC sorted from wild type animals from each developmental stage,,pubmed:30924555,,192CS1,GSM3397468,,source name:192hpf GFAP:GFP Negative|age:192hpf|genotype:Control GFAP:GFP|tissue:Dissociated retinal tissue|gfap:Negative,192CS1,Trimmomatic read trimming Hisat2 genome aligment Samtools Sam to Bam sorting Bioconuducter Feature counts  Rsubread  limma DESeq2  DEFormats  org.Dr.eg.db  EdgeR Genome build: GRCz10 Supplementary files format and content: TMM fold change analysis,192hpf GFAP:GFP Negative,,Single cell suspensions were sorted on a Beckman Coulter MoFlo to capture Muller glia GFP and control retinal tissue non GFP. Cells were sorted into lysis buffer  and RNA was immediately extracted using the RNeasy mini kit Qiagen. RNA concentration and qualities were assessed on an Agilent Bioanalyzer and RNA amplification and cDNA synthesis was performed with the Ovation RNA Amplification System V2 NuGEN using manufacturer’s protocol. Nextera library preparations were performed using the Nextera DNA library kit according to the manufacturer's directions,,age:192hpf|genotype:Control GFAP:GFP|tissue:Dissociated retinal tissue|gfap:Negative,GSM3397468,GSM3397468: 192CS1; Danio rerio; RNA Seq,GSM3397468,,1,Single cell suspensions were sorted on a Beckman Coulter MoFlo to capture Muller glia GFP and control retinal tissue non GFP. Cells were sorted into lysis buffer  and RNA was immediately extracted using the RNeasy mini kit Qiagen. RNA concentration and qualities were assessed on an Agilent Bioanalyzer and RNA amplification and cDNA synthesis was performed with the Ovation RNA Amplification System V2 NuGEN using manufacturer's protocol. Nextera library preparations were performed using the Nextera DNA library kit according to the manufacturer's directions,GEO Accession:GSM3397468,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2500,,SRP162288,,dangling references:treat as unmapped,192CS1.bam,bam,3097074000.0,24776592.0,GSM3397468 r1,0:125,A:802653377;C:765906943;G:684854694;T:843590152;N:68834,125,,,,802653377,765906943,684854694,843590152,68834,SRX4724286,SRS3809061,SRA780762,GEO,University of Cambridge,1,0.4094,,0.05897,,0.79754,,0.56363,,125,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,nextera,bulk,unknown,unknown,,United Kingdom,2018-09-20,Larval,Larval,Eye,Sensory System
49302,SRR7886633,SRX4724285,SRS3809059,SRP162288,PRJNA492363,Genetic control of cellular morphogenesis in Müller glia,GSE120275,Transcriptome Analysis,How the various cell types of the body achieve their specific shapes is fundamentally unknown.  Here  we explore this issue by identifying genes involved in the elaboration of the complex  yet conserved  cellular morphology of Müller glial MG cells in the retina.  Using genomic  based strategies in zebrafish  we found more than 40 candidate genes involved in specific aspects of MG morphogenesis.  The successive steps of cell morphogenesis correlate with the timing of the expression of cohorts of inter related genes that have roles in generating the particular anatomical features of these cells  suggesting that a sequence of genetic regulomes govern stepwise cellular morphogenesis in this system. Overall design: 12 samples with three replicates each are provided.  GFAP:GFP positive and negative cells were FAC sorted from wild type animals from each developmental stage,,pubmed:30924555,,120MGS3,GSM3397467,,source name:120hpf GFAP:GFP Positive|age:120hpf|genotype:Control GFAP:GFP|tissue:Dissociated retinal tissue|gfap:Positive,120MGS3,Trimmomatic read trimming Hisat2 genome aligment Samtools Sam to Bam sorting Bioconuducter Feature counts  Rsubread  limma DESeq2  DEFormats  org.Dr.eg.db  EdgeR Genome build: GRCz10 Supplementary files format and content: TMM fold change analysis,120hpf GFAP:GFP Positive,,Single cell suspensions were sorted on a Beckman Coulter MoFlo to capture Muller glia GFP and control retinal tissue non GFP. Cells were sorted into lysis buffer  and RNA was immediately extracted using the RNeasy mini kit Qiagen. RNA concentration and qualities were assessed on an Agilent Bioanalyzer and RNA amplification and cDNA synthesis was performed with the Ovation RNA Amplification System V2 NuGEN using manufacturer’s protocol. Nextera library preparations were performed using the Nextera DNA library kit according to the manufacturer's directions,,age:120hpf|genotype:Control GFAP:GFP|tissue:Dissociated retinal tissue|gfap:Positive,GSM3397467,GSM3397467: 120MGS3; Danio rerio; RNA Seq,GSM3397467,,1,Single cell suspensions were sorted on a Beckman Coulter MoFlo to capture Muller glia GFP and control retinal tissue non GFP. Cells were sorted into lysis buffer  and RNA was immediately extracted using the RNeasy mini kit Qiagen. RNA concentration and qualities were assessed on an Agilent Bioanalyzer and RNA amplification and cDNA synthesis was performed with the Ovation RNA Amplification System V2 NuGEN using manufacturer's protocol. Nextera library preparations were performed using the Nextera DNA library kit according to the manufacturer's directions,GEO Accession:GSM3397467,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2500,,SRP162288,,dangling references:treat as unmapped,120MGS3.bam,bam,2826290000.0,22610320.0,GSM3397467 r1,0:125,A:807896468;C:639830897;G:571446947;T:807097846;N:17842,125,,,,807896468,639830897,571446947,807097846,17842,SRX4724285,SRS3809059,SRA780762,GEO,University of Cambridge,1,0.82593,,0.48098,,0.87091,,0.46559,,125,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,nextera,bulk,unknown,unknown,,United Kingdom,2018-09-20,Larval,Larval,Eye,Sensory System
49303,SRR7886632,SRX4724284,SRS3809057,SRP162288,PRJNA492363,Genetic control of cellular morphogenesis in Müller glia,GSE120275,Transcriptome Analysis,How the various cell types of the body achieve their specific shapes is fundamentally unknown.  Here  we explore this issue by identifying genes involved in the elaboration of the complex  yet conserved  cellular morphology of Müller glial MG cells in the retina.  Using genomic  based strategies in zebrafish  we found more than 40 candidate genes involved in specific aspects of MG morphogenesis.  The successive steps of cell morphogenesis correlate with the timing of the expression of cohorts of inter related genes that have roles in generating the particular anatomical features of these cells  suggesting that a sequence of genetic regulomes govern stepwise cellular morphogenesis in this system. Overall design: 12 samples with three replicates each are provided.  GFAP:GFP positive and negative cells were FAC sorted from wild type animals from each developmental stage,,pubmed:30924555,,120MGS2,GSM3397466,,source name:120hpf GFAP:GFP Positive|age:120hpf|genotype:Control GFAP:GFP|tissue:Dissociated retinal tissue|gfap:Positive,120MGS2,Trimmomatic read trimming Hisat2 genome aligment Samtools Sam to Bam sorting Bioconuducter Feature counts  Rsubread  limma DESeq2  DEFormats  org.Dr.eg.db  EdgeR Genome build: GRCz10 Supplementary files format and content: TMM fold change analysis,120hpf GFAP:GFP Positive,,Single cell suspensions were sorted on a Beckman Coulter MoFlo to capture Muller glia GFP and control retinal tissue non GFP. Cells were sorted into lysis buffer  and RNA was immediately extracted using the RNeasy mini kit Qiagen. RNA concentration and qualities were assessed on an Agilent Bioanalyzer and RNA amplification and cDNA synthesis was performed with the Ovation RNA Amplification System V2 NuGEN using manufacturer’s protocol. Nextera library preparations were performed using the Nextera DNA library kit according to the manufacturer's directions,,age:120hpf|genotype:Control GFAP:GFP|tissue:Dissociated retinal tissue|gfap:Positive,GSM3397466,GSM3397466: 120MGS2; Danio rerio; RNA Seq,GSM3397466,,1,Single cell suspensions were sorted on a Beckman Coulter MoFlo to capture Muller glia GFP and control retinal tissue non GFP. Cells were sorted into lysis buffer  and RNA was immediately extracted using the RNeasy mini kit Qiagen. RNA concentration and qualities were assessed on an Agilent Bioanalyzer and RNA amplification and cDNA synthesis was performed with the Ovation RNA Amplification System V2 NuGEN using manufacturer's protocol. Nextera library preparations were performed using the Nextera DNA library kit according to the manufacturer's directions,GEO Accession:GSM3397466,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2500,,SRP162288,,dangling references:treat as unmapped,120MGS2.bam,bam,2957896875.0,23663175.0,GSM3397466 r1,0:125,A:844269581;C:636615963;G:626001648;T:850992847;N:16836,125,,,,844269581,636615963,626001648,850992847,16836,SRX4724284,SRS3809057,SRA780762,GEO,University of Cambridge,1,0.82727,,0.48182,,0.87493,,0.4699,,125,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,nextera,bulk,unknown,unknown,,United Kingdom,2018-09-20,Larval,Larval,Eye,Sensory System
49304,SRR7886631,SRX4724283,SRS3809058,SRP162288,PRJNA492363,Genetic control of cellular morphogenesis in Müller glia,GSE120275,Transcriptome Analysis,How the various cell types of the body achieve their specific shapes is fundamentally unknown.  Here  we explore this issue by identifying genes involved in the elaboration of the complex  yet conserved  cellular morphology of Müller glial MG cells in the retina.  Using genomic  based strategies in zebrafish  we found more than 40 candidate genes involved in specific aspects of MG morphogenesis.  The successive steps of cell morphogenesis correlate with the timing of the expression of cohorts of inter related genes that have roles in generating the particular anatomical features of these cells  suggesting that a sequence of genetic regulomes govern stepwise cellular morphogenesis in this system. Overall design: 12 samples with three replicates each are provided.  GFAP:GFP positive and negative cells were FAC sorted from wild type animals from each developmental stage,,pubmed:30924555,,120MGS1,GSM3397465,,source name:120hpf GFAP:GFP Positive|age:120hpf|genotype:Control GFAP:GFP|tissue:Dissociated retinal tissue|gfap:Positive,120MGS1,Trimmomatic read trimming Hisat2 genome aligment Samtools Sam to Bam sorting Bioconuducter Feature counts  Rsubread  limma DESeq2  DEFormats  org.Dr.eg.db  EdgeR Genome build: GRCz10 Supplementary files format and content: TMM fold change analysis,120hpf GFAP:GFP Positive,,Single cell suspensions were sorted on a Beckman Coulter MoFlo to capture Muller glia GFP and control retinal tissue non GFP. Cells were sorted into lysis buffer  and RNA was immediately extracted using the RNeasy mini kit Qiagen. RNA concentration and qualities were assessed on an Agilent Bioanalyzer and RNA amplification and cDNA synthesis was performed with the Ovation RNA Amplification System V2 NuGEN using manufacturer’s protocol. Nextera library preparations were performed using the Nextera DNA library kit according to the manufacturer's directions,,age:120hpf|genotype:Control GFAP:GFP|tissue:Dissociated retinal tissue|gfap:Positive,GSM3397465,GSM3397465: 120MGS1; Danio rerio; RNA Seq,GSM3397465,,1,Single cell suspensions were sorted on a Beckman Coulter MoFlo to capture Muller glia GFP and control retinal tissue non GFP. Cells were sorted into lysis buffer  and RNA was immediately extracted using the RNeasy mini kit Qiagen. RNA concentration and qualities were assessed on an Agilent Bioanalyzer and RNA amplification and cDNA synthesis was performed with the Ovation RNA Amplification System V2 NuGEN using manufacturer's protocol. Nextera library preparations were performed using the Nextera DNA library kit according to the manufacturer's directions,GEO Accession:GSM3397465,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2500,,SRP162288,,dangling references:treat as unmapped,120MGS1.bam,bam,2957896875.0,23663175.0,GSM3397465 r1,0:125,A:847629815;C:668284159;G:595326951;T:846591331;N:64619,125,,,,847629815,668284159,595326951,846591331,64619,SRX4724283,SRS3809058,SRA780762,GEO,University of Cambridge,1,0.82537,,0.47969,,0.87229,,0.47431,,125,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,nextera,bulk,unknown,unknown,,United Kingdom,2018-09-20,Larval,Larval,Eye,Sensory System