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 34206,SRR31539731,SRX26906755,SRS23386341,SRP548139,PRJNA1191617,Sox10 is required for systemic initiation of bone mineralization,GSE283071,Transcriptome Analysis,Heterozygous variants in SOX10 cause syndromes affecting pigmentation digestion hearing and neural development primarily attributable to failed differentiation or loss of non skeletal neural crest derivatives. We report here an additional novel requirement for Sox10 in bone mineralization. Neither crest nor mesoderm derived bones initiate mineralization on time in zebrafish sox10 mutants despite normal osteoblast differentiation and matrix production. Mutants are deficient in the Trpv6+ ionocytes that take up calcium from the environment resulting in severe calcium deficiency. As these ionocytes derive from ectoderm not crest we hypothesized that the primary defect resides in a separate organ that systemically regulates ionocyte numbers. RNAseq revealed significantly elevated stanniocalcin Stc1a an anti hypercalcemic hormone in sox10 mutants. Stc1a inhibits calcium uptake in fish by repressing trpv6 expression and Trpv6+ ionocyte proliferation. Epistasis assays confirm excess Stc1a as the proximate cause of the calcium deficit. The pronephros derived glands that synthesize Stc1a interact with sox10+ cells but these cells are missing in mutants. We conclude that sox10+ crest derived cells non autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary to initiate bone mineralization. Overall design: Bulk RNA seq was performed on pooled whole body zebrafish wild type and sox10 mutant larvae at 45 hpf 4 dpf and 7 dpf. Total of 18 samples. 45 hpf 3 replicates: controls n = 10 20 & 25; mutants n = 10 15 & 15 embryos 4 dpf 3 replicates: controls n = 10 15 & 12; mutants n = 10 11 & 12 embryos and 7 dpf 3 replicates: controls n = 12 30 & 18; mutants = 18 12 & 7 embryos,,pubmed:39791977,,Whole body 7 dpf sox10 mutant embryos biol rep 3,GSM8655460,,source name:whole body|tissue:whole body|genotype:sox10 / |time:7 dpf|geo loc name:missing|collection date:missing,Whole body 7 dpf sox10 mutant embryos biol rep 3,The data was processed through the Galaxy project using Trimmomatic version 0.39 Sequences were pseudoaligned and quantified at TPM values using Kallisto. Assembly: GRCz11 Supplementary files format and content: Tab delimited text file includes TPM values for each transcript at 45 hpf 4 dpf and 7 dpf,whole body,,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina’s TruSeq® stranded mRNA library preparation kit.,,tissue:whole body|genotype:sox10 / |time:7 dpf,GSM8655460,GSM8655460: Whole body 7 dpf sox10 mutant embryos biol rep 3; Danio rerio; RNA Seq,GSM8655460 r1,GSM8655460,1,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina's TruSeq® stranded mRNA library preparation kit.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP548139,,,7 dpf_mut 3_R1.fastq.gz 7 dpf_mut 3_R2.fastq.gz,fastq fastq,14265929756.0,47238178.0,GSM8655460 r1,0:151 1:151,A:3890017873;C:3282588566;G:3079614796;T:4012518928;N:1189593,151,151,,,3890017873,3282588566,3079614796,4012518928,1189593,SRX26906755,SRS23386341,,,"Barske Lab, Human Genetics, Cincinnati Children's Hospital",,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2024-11-27,Multi-stage,Multi-stage,Trunk,Surface Structure 34207,SRR31539732,SRX26906754,SRS23386340,SRP548139,PRJNA1191617,Sox10 is required for systemic initiation of bone mineralization,GSE283071,Transcriptome Analysis,Heterozygous variants in SOX10 cause syndromes affecting pigmentation digestion hearing and neural development primarily attributable to failed differentiation or loss of non skeletal neural crest derivatives. We report here an additional novel requirement for Sox10 in bone mineralization. Neither crest nor mesoderm derived bones initiate mineralization on time in zebrafish sox10 mutants despite normal osteoblast differentiation and matrix production. Mutants are deficient in the Trpv6+ ionocytes that take up calcium from the environment resulting in severe calcium deficiency. As these ionocytes derive from ectoderm not crest we hypothesized that the primary defect resides in a separate organ that systemically regulates ionocyte numbers. RNAseq revealed significantly elevated stanniocalcin Stc1a an anti hypercalcemic hormone in sox10 mutants. Stc1a inhibits calcium uptake in fish by repressing trpv6 expression and Trpv6+ ionocyte proliferation. Epistasis assays confirm excess Stc1a as the proximate cause of the calcium deficit. The pronephros derived glands that synthesize Stc1a interact with sox10+ cells but these cells are missing in mutants. We conclude that sox10+ crest derived cells non autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary to initiate bone mineralization. Overall design: Bulk RNA seq was performed on pooled whole body zebrafish wild type and sox10 mutant larvae at 45 hpf 4 dpf and 7 dpf. Total of 18 samples. 45 hpf 3 replicates: controls n = 10 20 & 25; mutants n = 10 15 & 15 embryos 4 dpf 3 replicates: controls n = 10 15 & 12; mutants n = 10 11 & 12 embryos and 7 dpf 3 replicates: controls n = 12 30 & 18; mutants = 18 12 & 7 embryos,,pubmed:39791977,,Whole body 7 dpf sox10 mutant embryos biol rep 2,GSM8655459,,source name:whole body|tissue:whole body|genotype:sox10 / |time:7 dpf|geo loc name:missing|collection date:missing,Whole body 7 dpf sox10 mutant embryos biol rep 2,The data was processed through the Galaxy project using Trimmomatic version 0.39 Sequences were pseudoaligned and quantified at TPM values using Kallisto. Assembly: GRCz11 Supplementary files format and content: Tab delimited text file includes TPM values for each transcript at 45 hpf 4 dpf and 7 dpf,whole body,,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina’s TruSeq® stranded mRNA library preparation kit.,,tissue:whole body|genotype:sox10 / |time:7 dpf,GSM8655459,GSM8655459: Whole body 7 dpf sox10 mutant embryos biol rep 2; Danio rerio; RNA Seq,GSM8655459 r1,GSM8655459,1,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina's TruSeq® stranded mRNA library preparation kit.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP548139,,,7 dpf_mut 2_R1.fastq.gz 7 dpf_mut 2_R2.fastq.gz,fastq fastq,16096968138.0,53301219.0,GSM8655459 r1,0:151 1:151,A:4158085994;C:3672102993;G:3915623401;T:4349806816;N:1348934,151,151,,,4158085994,3672102993,3915623401,4349806816,1348934,SRX26906754,SRS23386340,,,"Barske Lab, Human Genetics, Cincinnati Children's Hospital",,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2024-11-27,Multi-stage,Multi-stage,Trunk,Surface Structure 34208,SRR31539733,SRX26906753,SRS23386339,SRP548139,PRJNA1191617,Sox10 is required for systemic initiation of bone mineralization,GSE283071,Transcriptome Analysis,Heterozygous variants in SOX10 cause syndromes affecting pigmentation digestion hearing and neural development primarily attributable to failed differentiation or loss of non skeletal neural crest derivatives. We report here an additional novel requirement for Sox10 in bone mineralization. Neither crest nor mesoderm derived bones initiate mineralization on time in zebrafish sox10 mutants despite normal osteoblast differentiation and matrix production. Mutants are deficient in the Trpv6+ ionocytes that take up calcium from the environment resulting in severe calcium deficiency. As these ionocytes derive from ectoderm not crest we hypothesized that the primary defect resides in a separate organ that systemically regulates ionocyte numbers. RNAseq revealed significantly elevated stanniocalcin Stc1a an anti hypercalcemic hormone in sox10 mutants. Stc1a inhibits calcium uptake in fish by repressing trpv6 expression and Trpv6+ ionocyte proliferation. Epistasis assays confirm excess Stc1a as the proximate cause of the calcium deficit. The pronephros derived glands that synthesize Stc1a interact with sox10+ cells but these cells are missing in mutants. We conclude that sox10+ crest derived cells non autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary to initiate bone mineralization. Overall design: Bulk RNA seq was performed on pooled whole body zebrafish wild type and sox10 mutant larvae at 45 hpf 4 dpf and 7 dpf. Total of 18 samples. 45 hpf 3 replicates: controls n = 10 20 & 25; mutants n = 10 15 & 15 embryos 4 dpf 3 replicates: controls n = 10 15 & 12; mutants n = 10 11 & 12 embryos and 7 dpf 3 replicates: controls n = 12 30 & 18; mutants = 18 12 & 7 embryos,,pubmed:39791977,,Whole body 7 dpf sox10 mutant embryos biol rep 1,GSM8655458,,source name:whole body|tissue:whole body|genotype:sox10 / |time:7 dpf|geo loc name:missing|collection date:missing,Whole body 7 dpf sox10 mutant embryos biol rep 1,The data was processed through the Galaxy project using Trimmomatic version 0.39 Sequences were pseudoaligned and quantified at TPM values using Kallisto. Assembly: GRCz11 Supplementary files format and content: Tab delimited text file includes TPM values for each transcript at 45 hpf 4 dpf and 7 dpf,whole body,,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina’s TruSeq® stranded mRNA library preparation kit.,,tissue:whole body|genotype:sox10 / |time:7 dpf,GSM8655458,GSM8655458: Whole body 7 dpf sox10 mutant embryos biol rep 1; Danio rerio; RNA Seq,GSM8655458 r1,GSM8655458,1,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina's TruSeq® stranded mRNA library preparation kit.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP548139,,,7 dpf_mut 1_R1.fastq.gz 7 dpf_mut 1_R2.fastq.gz,fastq fastq,15919165034.0,52712467.0,GSM8655458 r1,0:151 1:151,A:4153549187;C:3626188288;G:3782139805;T:4355972996;N:1314758,151,151,,,4153549187,3626188288,3782139805,4355972996,1314758,SRX26906753,SRS23386339,,,"Barske Lab, Human Genetics, Cincinnati Children's Hospital",,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2024-11-27,Multi-stage,Multi-stage,Trunk,Surface Structure 34209,SRR31539734,SRX26906752,SRS23386338,SRP548139,PRJNA1191617,Sox10 is required for systemic initiation of bone mineralization,GSE283071,Transcriptome Analysis,Heterozygous variants in SOX10 cause syndromes affecting pigmentation digestion hearing and neural development primarily attributable to failed differentiation or loss of non skeletal neural crest derivatives. We report here an additional novel requirement for Sox10 in bone mineralization. Neither crest nor mesoderm derived bones initiate mineralization on time in zebrafish sox10 mutants despite normal osteoblast differentiation and matrix production. Mutants are deficient in the Trpv6+ ionocytes that take up calcium from the environment resulting in severe calcium deficiency. As these ionocytes derive from ectoderm not crest we hypothesized that the primary defect resides in a separate organ that systemically regulates ionocyte numbers. RNAseq revealed significantly elevated stanniocalcin Stc1a an anti hypercalcemic hormone in sox10 mutants. Stc1a inhibits calcium uptake in fish by repressing trpv6 expression and Trpv6+ ionocyte proliferation. Epistasis assays confirm excess Stc1a as the proximate cause of the calcium deficit. The pronephros derived glands that synthesize Stc1a interact with sox10+ cells but these cells are missing in mutants. We conclude that sox10+ crest derived cells non autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary to initiate bone mineralization. Overall design: Bulk RNA seq was performed on pooled whole body zebrafish wild type and sox10 mutant larvae at 45 hpf 4 dpf and 7 dpf. Total of 18 samples. 45 hpf 3 replicates: controls n = 10 20 & 25; mutants n = 10 15 & 15 embryos 4 dpf 3 replicates: controls n = 10 15 & 12; mutants n = 10 11 & 12 embryos and 7 dpf 3 replicates: controls n = 12 30 & 18; mutants = 18 12 & 7 embryos,,pubmed:39791977,,Whole body 7 dpf wild type embryos biol rep 3,GSM8655457,,source name:whole body|tissue:whole body|genotype:WT|time:7 dpf|geo loc name:missing|collection date:missing,Whole body 7 dpf wild type embryos biol rep 3,The data was processed through the Galaxy project using Trimmomatic version 0.39 Sequences were pseudoaligned and quantified at TPM values using Kallisto. Assembly: GRCz11 Supplementary files format and content: Tab delimited text file includes TPM values for each transcript at 45 hpf 4 dpf and 7 dpf,whole body,,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina’s TruSeq® stranded mRNA library preparation kit.,,tissue:whole body|genotype:WT|time:7 dpf,GSM8655457,GSM8655457: Whole body 7 dpf wild type embryos biol rep 3; Danio rerio; RNA Seq,GSM8655457 r1,GSM8655457,1,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina's TruSeq® stranded mRNA library preparation kit.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP548139,,,7 dpf_WT 3_R1.fastq.gz 7 dpf_WT 3_R2.fastq.gz,fastq fastq,15543953590.0,51470045.0,GSM8655457 r1,0:151 1:151,A:4031818994;C:3704599213;G:3577251498;T:4228986622;N:1297263,151,151,,,4031818994,3704599213,3577251498,4228986622,1297263,SRX26906752,SRS23386338,,,"Barske Lab, Human Genetics, Cincinnati Children's Hospital",,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2024-11-27,Multi-stage,Multi-stage,Trunk,Surface Structure 34210,SRR31539735,SRX26906751,SRS23386337,SRP548139,PRJNA1191617,Sox10 is required for systemic initiation of bone mineralization,GSE283071,Transcriptome Analysis,Heterozygous variants in SOX10 cause syndromes affecting pigmentation digestion hearing and neural development primarily attributable to failed differentiation or loss of non skeletal neural crest derivatives. We report here an additional novel requirement for Sox10 in bone mineralization. Neither crest nor mesoderm derived bones initiate mineralization on time in zebrafish sox10 mutants despite normal osteoblast differentiation and matrix production. Mutants are deficient in the Trpv6+ ionocytes that take up calcium from the environment resulting in severe calcium deficiency. As these ionocytes derive from ectoderm not crest we hypothesized that the primary defect resides in a separate organ that systemically regulates ionocyte numbers. RNAseq revealed significantly elevated stanniocalcin Stc1a an anti hypercalcemic hormone in sox10 mutants. Stc1a inhibits calcium uptake in fish by repressing trpv6 expression and Trpv6+ ionocyte proliferation. Epistasis assays confirm excess Stc1a as the proximate cause of the calcium deficit. The pronephros derived glands that synthesize Stc1a interact with sox10+ cells but these cells are missing in mutants. We conclude that sox10+ crest derived cells non autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary to initiate bone mineralization. Overall design: Bulk RNA seq was performed on pooled whole body zebrafish wild type and sox10 mutant larvae at 45 hpf 4 dpf and 7 dpf. Total of 18 samples. 45 hpf 3 replicates: controls n = 10 20 & 25; mutants n = 10 15 & 15 embryos 4 dpf 3 replicates: controls n = 10 15 & 12; mutants n = 10 11 & 12 embryos and 7 dpf 3 replicates: controls n = 12 30 & 18; mutants = 18 12 & 7 embryos,,pubmed:39791977,,Whole body 7 dpf wild type embryos biol rep 2,GSM8655456,,source name:whole body|tissue:whole body|genotype:WT|time:7 dpf|geo loc name:missing|collection date:missing,Whole body 7 dpf wild type embryos biol rep 2,The data was processed through the Galaxy project using Trimmomatic version 0.39 Sequences were pseudoaligned and quantified at TPM values using Kallisto. Assembly: GRCz11 Supplementary files format and content: Tab delimited text file includes TPM values for each transcript at 45 hpf 4 dpf and 7 dpf,whole body,,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina’s TruSeq® stranded mRNA library preparation kit.,,tissue:whole body|genotype:WT|time:7 dpf,GSM8655456,GSM8655456: Whole body 7 dpf wild type embryos biol rep 2; Danio rerio; RNA Seq,GSM8655456 r1,GSM8655456,1,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina's TruSeq® stranded mRNA library preparation kit.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP548139,,,7 dpf_WT 2_R1.fastq.gz 7 dpf_WT 2_R2.fastq.gz,fastq fastq,14325089744.0,47434072.0,GSM8655456 r1,0:151 1:151,A:3806636861;C:3343198938;G:3185927363;T:3988104951;N:1221631,151,151,,,3806636861,3343198938,3185927363,3988104951,1221631,SRX26906751,SRS23386337,,,"Barske Lab, Human Genetics, Cincinnati Children's Hospital",,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2024-11-27,Multi-stage,Multi-stage,Trunk,Surface Structure 34211,SRR31539736,SRX26906750,SRS23386333,SRP548139,PRJNA1191617,Sox10 is required for systemic initiation of bone mineralization,GSE283071,Transcriptome Analysis,Heterozygous variants in SOX10 cause syndromes affecting pigmentation digestion hearing and neural development primarily attributable to failed differentiation or loss of non skeletal neural crest derivatives. We report here an additional novel requirement for Sox10 in bone mineralization. Neither crest nor mesoderm derived bones initiate mineralization on time in zebrafish sox10 mutants despite normal osteoblast differentiation and matrix production. Mutants are deficient in the Trpv6+ ionocytes that take up calcium from the environment resulting in severe calcium deficiency. As these ionocytes derive from ectoderm not crest we hypothesized that the primary defect resides in a separate organ that systemically regulates ionocyte numbers. RNAseq revealed significantly elevated stanniocalcin Stc1a an anti hypercalcemic hormone in sox10 mutants. Stc1a inhibits calcium uptake in fish by repressing trpv6 expression and Trpv6+ ionocyte proliferation. Epistasis assays confirm excess Stc1a as the proximate cause of the calcium deficit. The pronephros derived glands that synthesize Stc1a interact with sox10+ cells but these cells are missing in mutants. We conclude that sox10+ crest derived cells non autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary to initiate bone mineralization. Overall design: Bulk RNA seq was performed on pooled whole body zebrafish wild type and sox10 mutant larvae at 45 hpf 4 dpf and 7 dpf. Total of 18 samples. 45 hpf 3 replicates: controls n = 10 20 & 25; mutants n = 10 15 & 15 embryos 4 dpf 3 replicates: controls n = 10 15 & 12; mutants n = 10 11 & 12 embryos and 7 dpf 3 replicates: controls n = 12 30 & 18; mutants = 18 12 & 7 embryos,,pubmed:39791977,,Whole body 7 dpf wild type embryos biol rep 1,GSM8655455,,source name:whole body|tissue:whole body|genotype:WT|time:7 dpf|geo loc name:missing|collection date:missing,Whole body 7 dpf wild type embryos biol rep 1,The data was processed through the Galaxy project using Trimmomatic version 0.39 Sequences were pseudoaligned and quantified at TPM values using Kallisto. Assembly: GRCz11 Supplementary files format and content: Tab delimited text file includes TPM values for each transcript at 45 hpf 4 dpf and 7 dpf,whole body,,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina’s TruSeq® stranded mRNA library preparation kit.,,tissue:whole body|genotype:WT|time:7 dpf,GSM8655455,GSM8655455: Whole body 7 dpf wild type embryos biol rep 1; Danio rerio; RNA Seq,GSM8655455 r1,GSM8655455,1,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina's TruSeq® stranded mRNA library preparation kit.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP548139,,,7 dpf_WT 1_R1.fastq.gz 7 dpf_WT 1_R2.fastq.gz,fastq fastq,16398118008.0,54298404.0,GSM8655455 r1,0:151 1:151,A:4341778579;C:3858758690;G:3657590940;T:4538608543;N:1381256,151,151,,,4341778579,3858758690,3657590940,4538608543,1381256,SRX26906750,SRS23386333,,,"Barske Lab, Human Genetics, Cincinnati Children's Hospital",,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2024-11-27,Multi-stage,Multi-stage,Trunk,Surface Structure 34212,SRR31539737,SRX26906749,SRS23386334,SRP548139,PRJNA1191617,Sox10 is required for systemic initiation of bone mineralization,GSE283071,Transcriptome Analysis,Heterozygous variants in SOX10 cause syndromes affecting pigmentation digestion hearing and neural development primarily attributable to failed differentiation or loss of non skeletal neural crest derivatives. We report here an additional novel requirement for Sox10 in bone mineralization. Neither crest nor mesoderm derived bones initiate mineralization on time in zebrafish sox10 mutants despite normal osteoblast differentiation and matrix production. Mutants are deficient in the Trpv6+ ionocytes that take up calcium from the environment resulting in severe calcium deficiency. As these ionocytes derive from ectoderm not crest we hypothesized that the primary defect resides in a separate organ that systemically regulates ionocyte numbers. RNAseq revealed significantly elevated stanniocalcin Stc1a an anti hypercalcemic hormone in sox10 mutants. Stc1a inhibits calcium uptake in fish by repressing trpv6 expression and Trpv6+ ionocyte proliferation. Epistasis assays confirm excess Stc1a as the proximate cause of the calcium deficit. The pronephros derived glands that synthesize Stc1a interact with sox10+ cells but these cells are missing in mutants. We conclude that sox10+ crest derived cells non autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary to initiate bone mineralization. Overall design: Bulk RNA seq was performed on pooled whole body zebrafish wild type and sox10 mutant larvae at 45 hpf 4 dpf and 7 dpf. Total of 18 samples. 45 hpf 3 replicates: controls n = 10 20 & 25; mutants n = 10 15 & 15 embryos 4 dpf 3 replicates: controls n = 10 15 & 12; mutants n = 10 11 & 12 embryos and 7 dpf 3 replicates: controls n = 12 30 & 18; mutants = 18 12 & 7 embryos,,pubmed:39791977,,Whole body 4 dpf sox10 mutant embryos biol rep 3,GSM8655454,,source name:whole body|tissue:whole body|genotype:sox10 / |time:4 dpf|geo loc name:missing|collection date:missing,Whole body 4 dpf sox10 mutant embryos biol rep 3,The data was processed through the Galaxy project using Trimmomatic version 0.39 Sequences were pseudoaligned and quantified at TPM values using Kallisto. Assembly: GRCz11 Supplementary files format and content: Tab delimited text file includes TPM values for each transcript at 45 hpf 4 dpf and 7 dpf,whole body,,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina’s TruSeq® stranded mRNA library preparation kit.,,tissue:whole body|genotype:sox10 / |time:4 dpf,GSM8655454,GSM8655454: Whole body 4 dpf sox10 mutant embryos biol rep 3; Danio rerio; RNA Seq,GSM8655454 r1,GSM8655454,1,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina's TruSeq® stranded mRNA library preparation kit.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP548139,,,4 dpf_mut 3_R1.fastq.gz 4 dpf_mut 3_R2.fastq.gz,fastq fastq,15071278894.0,49904897.0,GSM8655454 r1,0:151 1:151,A:3996223512;C:3537174277;G:3398338576;T:4138304072;N:1238457,151,151,,,3996223512,3537174277,3398338576,4138304072,1238457,SRX26906749,SRS23386334,,,"Barske Lab, Human Genetics, Cincinnati Children's Hospital",,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2024-11-27,Multi-stage,Multi-stage,Trunk,Surface Structure 34213,SRR31539738,SRX26906748,SRS23386336,SRP548139,PRJNA1191617,Sox10 is required for systemic initiation of bone mineralization,GSE283071,Transcriptome Analysis,Heterozygous variants in SOX10 cause syndromes affecting pigmentation digestion hearing and neural development primarily attributable to failed differentiation or loss of non skeletal neural crest derivatives. We report here an additional novel requirement for Sox10 in bone mineralization. Neither crest nor mesoderm derived bones initiate mineralization on time in zebrafish sox10 mutants despite normal osteoblast differentiation and matrix production. Mutants are deficient in the Trpv6+ ionocytes that take up calcium from the environment resulting in severe calcium deficiency. As these ionocytes derive from ectoderm not crest we hypothesized that the primary defect resides in a separate organ that systemically regulates ionocyte numbers. RNAseq revealed significantly elevated stanniocalcin Stc1a an anti hypercalcemic hormone in sox10 mutants. Stc1a inhibits calcium uptake in fish by repressing trpv6 expression and Trpv6+ ionocyte proliferation. Epistasis assays confirm excess Stc1a as the proximate cause of the calcium deficit. The pronephros derived glands that synthesize Stc1a interact with sox10+ cells but these cells are missing in mutants. We conclude that sox10+ crest derived cells non autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary to initiate bone mineralization. Overall design: Bulk RNA seq was performed on pooled whole body zebrafish wild type and sox10 mutant larvae at 45 hpf 4 dpf and 7 dpf. Total of 18 samples. 45 hpf 3 replicates: controls n = 10 20 & 25; mutants n = 10 15 & 15 embryos 4 dpf 3 replicates: controls n = 10 15 & 12; mutants n = 10 11 & 12 embryos and 7 dpf 3 replicates: controls n = 12 30 & 18; mutants = 18 12 & 7 embryos,,pubmed:39791977,,Whole body 4 dpf sox10 mutant embryos biol rep 2,GSM8655453,,source name:whole body|tissue:whole body|genotype:sox10 / |time:4 dpf|geo loc name:missing|collection date:missing,Whole body 4 dpf sox10 mutant embryos biol rep 2,The data was processed through the Galaxy project using Trimmomatic version 0.39 Sequences were pseudoaligned and quantified at TPM values using Kallisto. Assembly: GRCz11 Supplementary files format and content: Tab delimited text file includes TPM values for each transcript at 45 hpf 4 dpf and 7 dpf,whole body,,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina’s TruSeq® stranded mRNA library preparation kit.,,tissue:whole body|genotype:sox10 / |time:4 dpf,GSM8655453,GSM8655453: Whole body 4 dpf sox10 mutant embryos biol rep 2; Danio rerio; RNA Seq,GSM8655453 r1,GSM8655453,1,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina's TruSeq® stranded mRNA library preparation kit.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP548139,,,4 dpf_mut 2_R1.fastq.gz 4 dpf_mut 2_R2.fastq.gz,fastq fastq,14918687958.0,49399629.0,GSM8655453 r1,0:151 1:151,A:3994132161;C:3464356999;G:3293001897;T:4165943265;N:1253636,151,151,,,3994132161,3464356999,3293001897,4165943265,1253636,SRX26906748,SRS23386336,,,"Barske Lab, Human Genetics, Cincinnati Children's Hospital",,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2024-11-27,Multi-stage,Multi-stage,Trunk,Surface Structure 34214,SRR31539739,SRX26906747,SRS23386332,SRP548139,PRJNA1191617,Sox10 is required for systemic initiation of bone mineralization,GSE283071,Transcriptome Analysis,Heterozygous variants in SOX10 cause syndromes affecting pigmentation digestion hearing and neural development primarily attributable to failed differentiation or loss of non skeletal neural crest derivatives. We report here an additional novel requirement for Sox10 in bone mineralization. Neither crest nor mesoderm derived bones initiate mineralization on time in zebrafish sox10 mutants despite normal osteoblast differentiation and matrix production. Mutants are deficient in the Trpv6+ ionocytes that take up calcium from the environment resulting in severe calcium deficiency. As these ionocytes derive from ectoderm not crest we hypothesized that the primary defect resides in a separate organ that systemically regulates ionocyte numbers. RNAseq revealed significantly elevated stanniocalcin Stc1a an anti hypercalcemic hormone in sox10 mutants. Stc1a inhibits calcium uptake in fish by repressing trpv6 expression and Trpv6+ ionocyte proliferation. Epistasis assays confirm excess Stc1a as the proximate cause of the calcium deficit. The pronephros derived glands that synthesize Stc1a interact with sox10+ cells but these cells are missing in mutants. We conclude that sox10+ crest derived cells non autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary to initiate bone mineralization. Overall design: Bulk RNA seq was performed on pooled whole body zebrafish wild type and sox10 mutant larvae at 45 hpf 4 dpf and 7 dpf. Total of 18 samples. 45 hpf 3 replicates: controls n = 10 20 & 25; mutants n = 10 15 & 15 embryos 4 dpf 3 replicates: controls n = 10 15 & 12; mutants n = 10 11 & 12 embryos and 7 dpf 3 replicates: controls n = 12 30 & 18; mutants = 18 12 & 7 embryos,,pubmed:39791977,,Whole body 4 dpf sox10 mutant embryos biol rep 1,GSM8655452,,source name:whole body|tissue:whole body|genotype:sox10 / |time:4 dpf|geo loc name:missing|collection date:missing,Whole body 4 dpf sox10 mutant embryos biol rep 1,The data was processed through the Galaxy project using Trimmomatic version 0.39 Sequences were pseudoaligned and quantified at TPM values using Kallisto. Assembly: GRCz11 Supplementary files format and content: Tab delimited text file includes TPM values for each transcript at 45 hpf 4 dpf and 7 dpf,whole body,,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina’s TruSeq® stranded mRNA library preparation kit.,,tissue:whole body|genotype:sox10 / |time:4 dpf,GSM8655452,GSM8655452: Whole body 4 dpf sox10 mutant embryos biol rep 1; Danio rerio; RNA Seq,GSM8655452 r1,GSM8655452,1,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina's TruSeq® stranded mRNA library preparation kit.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP548139,,,4 dpf_mut 1_R1.fastq.gz 4 dpf_mut 1_R2.fastq.gz,fastq fastq,14573247372.0,48255786.0,GSM8655452 r1,0:151 1:151,A:3964700989;C:3340575136;G:3128233836;T:4138502375;N:1235036,151,151,,,3964700989,3340575136,3128233836,4138502375,1235036,SRX26906747,SRS23386332,,,"Barske Lab, Human Genetics, Cincinnati Children's Hospital",,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2024-11-27,Multi-stage,Multi-stage,Trunk,Surface Structure 34215,SRR31539740,SRX26906746,SRS23386331,SRP548139,PRJNA1191617,Sox10 is required for systemic initiation of bone mineralization,GSE283071,Transcriptome Analysis,Heterozygous variants in SOX10 cause syndromes affecting pigmentation digestion hearing and neural development primarily attributable to failed differentiation or loss of non skeletal neural crest derivatives. We report here an additional novel requirement for Sox10 in bone mineralization. Neither crest nor mesoderm derived bones initiate mineralization on time in zebrafish sox10 mutants despite normal osteoblast differentiation and matrix production. Mutants are deficient in the Trpv6+ ionocytes that take up calcium from the environment resulting in severe calcium deficiency. As these ionocytes derive from ectoderm not crest we hypothesized that the primary defect resides in a separate organ that systemically regulates ionocyte numbers. RNAseq revealed significantly elevated stanniocalcin Stc1a an anti hypercalcemic hormone in sox10 mutants. Stc1a inhibits calcium uptake in fish by repressing trpv6 expression and Trpv6+ ionocyte proliferation. Epistasis assays confirm excess Stc1a as the proximate cause of the calcium deficit. The pronephros derived glands that synthesize Stc1a interact with sox10+ cells but these cells are missing in mutants. We conclude that sox10+ crest derived cells non autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary to initiate bone mineralization. Overall design: Bulk RNA seq was performed on pooled whole body zebrafish wild type and sox10 mutant larvae at 45 hpf 4 dpf and 7 dpf. Total of 18 samples. 45 hpf 3 replicates: controls n = 10 20 & 25; mutants n = 10 15 & 15 embryos 4 dpf 3 replicates: controls n = 10 15 & 12; mutants n = 10 11 & 12 embryos and 7 dpf 3 replicates: controls n = 12 30 & 18; mutants = 18 12 & 7 embryos,,pubmed:39791977,,Whole body 4 dpf wild type embryos biol rep 3,GSM8655451,,source name:whole body|tissue:whole body|genotype:WT|time:4 dpf|geo loc name:missing|collection date:missing,Whole body 4 dpf wild type embryos biol rep 3,The data was processed through the Galaxy project using Trimmomatic version 0.39 Sequences were pseudoaligned and quantified at TPM values using Kallisto. Assembly: GRCz11 Supplementary files format and content: Tab delimited text file includes TPM values for each transcript at 45 hpf 4 dpf and 7 dpf,whole body,,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina’s TruSeq® stranded mRNA library preparation kit.,,tissue:whole body|genotype:WT|time:4 dpf,GSM8655451,GSM8655451: Whole body 4 dpf wild type embryos biol rep 3; Danio rerio; RNA Seq,GSM8655451 r1,GSM8655451,1,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina's TruSeq® stranded mRNA library preparation kit.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP548139,,,4 dpf_WT 3_R1.fastq.gz 4 dpf_WT 3_R2.fastq.gz,fastq fastq,15132219172.0,50106686.0,GSM8655451 r1,0:151 1:151,A:4030654907;C:3519071260;G:3404455031;T:4176756699;N:1281275,151,151,,,4030654907,3519071260,3404455031,4176756699,1281275,SRX26906746,SRS23386331,,,"Barske Lab, Human Genetics, Cincinnati Children's Hospital",,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2024-11-27,Multi-stage,Multi-stage,Trunk,Surface Structure 34216,SRR31539741,SRX26906745,SRS23386335,SRP548139,PRJNA1191617,Sox10 is required for systemic initiation of bone mineralization,GSE283071,Transcriptome Analysis,Heterozygous variants in SOX10 cause syndromes affecting pigmentation digestion hearing and neural development primarily attributable to failed differentiation or loss of non skeletal neural crest derivatives. We report here an additional novel requirement for Sox10 in bone mineralization. Neither crest nor mesoderm derived bones initiate mineralization on time in zebrafish sox10 mutants despite normal osteoblast differentiation and matrix production. Mutants are deficient in the Trpv6+ ionocytes that take up calcium from the environment resulting in severe calcium deficiency. As these ionocytes derive from ectoderm not crest we hypothesized that the primary defect resides in a separate organ that systemically regulates ionocyte numbers. RNAseq revealed significantly elevated stanniocalcin Stc1a an anti hypercalcemic hormone in sox10 mutants. Stc1a inhibits calcium uptake in fish by repressing trpv6 expression and Trpv6+ ionocyte proliferation. Epistasis assays confirm excess Stc1a as the proximate cause of the calcium deficit. The pronephros derived glands that synthesize Stc1a interact with sox10+ cells but these cells are missing in mutants. We conclude that sox10+ crest derived cells non autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary to initiate bone mineralization. Overall design: Bulk RNA seq was performed on pooled whole body zebrafish wild type and sox10 mutant larvae at 45 hpf 4 dpf and 7 dpf. Total of 18 samples. 45 hpf 3 replicates: controls n = 10 20 & 25; mutants n = 10 15 & 15 embryos 4 dpf 3 replicates: controls n = 10 15 & 12; mutants n = 10 11 & 12 embryos and 7 dpf 3 replicates: controls n = 12 30 & 18; mutants = 18 12 & 7 embryos,,pubmed:39791977,,Whole body 4 dpf wild type embryos biol rep 2,GSM8655450,,source name:whole body|tissue:whole body|genotype:WT|time:4 dpf|geo loc name:missing|collection date:missing,Whole body 4 dpf wild type embryos biol rep 2,The data was processed through the Galaxy project using Trimmomatic version 0.39 Sequences were pseudoaligned and quantified at TPM values using Kallisto. Assembly: GRCz11 Supplementary files format and content: Tab delimited text file includes TPM values for each transcript at 45 hpf 4 dpf and 7 dpf,whole body,,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina’s TruSeq® stranded mRNA library preparation kit.,,tissue:whole body|genotype:WT|time:4 dpf,GSM8655450,GSM8655450: Whole body 4 dpf wild type embryos biol rep 2; Danio rerio; RNA Seq,GSM8655450 r1,GSM8655450,1,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina's TruSeq® stranded mRNA library preparation kit.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP548139,,,4 dpf_WT 2_R1.fastq.gz 4 dpf_WT 2_R2.fastq.gz,fastq fastq,15546478914.0,51478407.0,GSM8655450 r1,0:151 1:151,A:4105803894;C:3606318412;G:3589944905;T:4243130619;N:1281084,151,151,,,4105803894,3606318412,3589944905,4243130619,1281084,SRX26906745,SRS23386335,,,"Barske Lab, Human Genetics, Cincinnati Children's Hospital",,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2024-11-27,Multi-stage,Multi-stage,Trunk,Surface Structure 34217,SRR31539742,SRX26906744,SRS23386330,SRP548139,PRJNA1191617,Sox10 is required for systemic initiation of bone mineralization,GSE283071,Transcriptome Analysis,Heterozygous variants in SOX10 cause syndromes affecting pigmentation digestion hearing and neural development primarily attributable to failed differentiation or loss of non skeletal neural crest derivatives. We report here an additional novel requirement for Sox10 in bone mineralization. Neither crest nor mesoderm derived bones initiate mineralization on time in zebrafish sox10 mutants despite normal osteoblast differentiation and matrix production. Mutants are deficient in the Trpv6+ ionocytes that take up calcium from the environment resulting in severe calcium deficiency. As these ionocytes derive from ectoderm not crest we hypothesized that the primary defect resides in a separate organ that systemically regulates ionocyte numbers. RNAseq revealed significantly elevated stanniocalcin Stc1a an anti hypercalcemic hormone in sox10 mutants. Stc1a inhibits calcium uptake in fish by repressing trpv6 expression and Trpv6+ ionocyte proliferation. Epistasis assays confirm excess Stc1a as the proximate cause of the calcium deficit. The pronephros derived glands that synthesize Stc1a interact with sox10+ cells but these cells are missing in mutants. We conclude that sox10+ crest derived cells non autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary to initiate bone mineralization. Overall design: Bulk RNA seq was performed on pooled whole body zebrafish wild type and sox10 mutant larvae at 45 hpf 4 dpf and 7 dpf. Total of 18 samples. 45 hpf 3 replicates: controls n = 10 20 & 25; mutants n = 10 15 & 15 embryos 4 dpf 3 replicates: controls n = 10 15 & 12; mutants n = 10 11 & 12 embryos and 7 dpf 3 replicates: controls n = 12 30 & 18; mutants = 18 12 & 7 embryos,,pubmed:39791977,,Whole body 4 dpf wild type embryos biol rep 1,GSM8655449,,source name:whole body|tissue:whole body|genotype:WT|time:4 dpf|geo loc name:missing|collection date:missing,Whole body 4 dpf wild type embryos biol rep 1,The data was processed through the Galaxy project using Trimmomatic version 0.39 Sequences were pseudoaligned and quantified at TPM values using Kallisto. Assembly: GRCz11 Supplementary files format and content: Tab delimited text file includes TPM values for each transcript at 45 hpf 4 dpf and 7 dpf,whole body,,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina’s TruSeq® stranded mRNA library preparation kit.,,tissue:whole body|genotype:WT|time:4 dpf,GSM8655449,GSM8655449: Whole body 4 dpf wild type embryos biol rep 1; Danio rerio; RNA Seq,GSM8655449 r1,GSM8655449,1,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina's TruSeq® stranded mRNA library preparation kit.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP548139,,,4 dpf_WT 1_R1.fastq.gz 4 dpf_WT 1_R2.fastq.gz,fastq fastq,15246602880.0,50485440.0,GSM8655449 r1,0:151 1:151,A:4017771448;C:3536729749;G:3495956173;T:4194910365;N:1235145,151,151,,,4017771448,3536729749,3495956173,4194910365,1235145,SRX26906744,SRS23386330,,,"Barske Lab, Human Genetics, Cincinnati Children's Hospital",,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2024-11-27,Multi-stage,Multi-stage,Trunk,Surface Structure 34218,SRR31539743,SRX26906743,SRS23386329,SRP548139,PRJNA1191617,Sox10 is required for systemic initiation of bone mineralization,GSE283071,Transcriptome Analysis,Heterozygous variants in SOX10 cause syndromes affecting pigmentation digestion hearing and neural development primarily attributable to failed differentiation or loss of non skeletal neural crest derivatives. We report here an additional novel requirement for Sox10 in bone mineralization. Neither crest nor mesoderm derived bones initiate mineralization on time in zebrafish sox10 mutants despite normal osteoblast differentiation and matrix production. Mutants are deficient in the Trpv6+ ionocytes that take up calcium from the environment resulting in severe calcium deficiency. As these ionocytes derive from ectoderm not crest we hypothesized that the primary defect resides in a separate organ that systemically regulates ionocyte numbers. RNAseq revealed significantly elevated stanniocalcin Stc1a an anti hypercalcemic hormone in sox10 mutants. Stc1a inhibits calcium uptake in fish by repressing trpv6 expression and Trpv6+ ionocyte proliferation. Epistasis assays confirm excess Stc1a as the proximate cause of the calcium deficit. The pronephros derived glands that synthesize Stc1a interact with sox10+ cells but these cells are missing in mutants. We conclude that sox10+ crest derived cells non autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary to initiate bone mineralization. Overall design: Bulk RNA seq was performed on pooled whole body zebrafish wild type and sox10 mutant larvae at 45 hpf 4 dpf and 7 dpf. Total of 18 samples. 45 hpf 3 replicates: controls n = 10 20 & 25; mutants n = 10 15 & 15 embryos 4 dpf 3 replicates: controls n = 10 15 & 12; mutants n = 10 11 & 12 embryos and 7 dpf 3 replicates: controls n = 12 30 & 18; mutants = 18 12 & 7 embryos,,pubmed:39791977,,Whole body 45 hpf sox10 mutant embryos biol rep 3,GSM8655448,,source name:whole body|tissue:whole body|genotype:sox10 / |time:45 hpf|geo loc name:missing|collection date:missing,Whole body 45 hpf sox10 mutant embryos biol rep 3,The data was processed through the Galaxy project using Trimmomatic version 0.39 Sequences were pseudoaligned and quantified at TPM values using Kallisto. Assembly: GRCz11 Supplementary files format and content: Tab delimited text file includes TPM values for each transcript at 45 hpf 4 dpf and 7 dpf,whole body,,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina’s TruSeq® stranded mRNA library preparation kit.,,tissue:whole body|genotype:sox10 / |time:45 hpf,GSM8655448,GSM8655448: Whole body 45 hpf sox10 mutant embryos biol rep 3; Danio rerio; RNA Seq,GSM8655448 r1,GSM8655448,1,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina's TruSeq® stranded mRNA library preparation kit.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP548139,,,45 hpf_mut 3_R1.fastq.gz 45 hpf_mut 3_R2.fastq.gz,fastq fastq,14338321572.0,47477886.0,GSM8655448 r1,0:151 1:151,A:3808719246;C:3382555065;G:3212075099;T:3933758367;N:1213795,151,151,,,3808719246,3382555065,3212075099,3933758367,1213795,SRX26906743,SRS23386329,,,"Barske Lab, Human Genetics, Cincinnati Children's Hospital",,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2024-11-27,Multi-stage,Multi-stage,Trunk,Surface Structure 34219,SRR31539744,SRX26906742,SRS23386326,SRP548139,PRJNA1191617,Sox10 is required for systemic initiation of bone mineralization,GSE283071,Transcriptome Analysis,Heterozygous variants in SOX10 cause syndromes affecting pigmentation digestion hearing and neural development primarily attributable to failed differentiation or loss of non skeletal neural crest derivatives. We report here an additional novel requirement for Sox10 in bone mineralization. Neither crest nor mesoderm derived bones initiate mineralization on time in zebrafish sox10 mutants despite normal osteoblast differentiation and matrix production. Mutants are deficient in the Trpv6+ ionocytes that take up calcium from the environment resulting in severe calcium deficiency. As these ionocytes derive from ectoderm not crest we hypothesized that the primary defect resides in a separate organ that systemically regulates ionocyte numbers. RNAseq revealed significantly elevated stanniocalcin Stc1a an anti hypercalcemic hormone in sox10 mutants. Stc1a inhibits calcium uptake in fish by repressing trpv6 expression and Trpv6+ ionocyte proliferation. Epistasis assays confirm excess Stc1a as the proximate cause of the calcium deficit. The pronephros derived glands that synthesize Stc1a interact with sox10+ cells but these cells are missing in mutants. We conclude that sox10+ crest derived cells non autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary to initiate bone mineralization. Overall design: Bulk RNA seq was performed on pooled whole body zebrafish wild type and sox10 mutant larvae at 45 hpf 4 dpf and 7 dpf. Total of 18 samples. 45 hpf 3 replicates: controls n = 10 20 & 25; mutants n = 10 15 & 15 embryos 4 dpf 3 replicates: controls n = 10 15 & 12; mutants n = 10 11 & 12 embryos and 7 dpf 3 replicates: controls n = 12 30 & 18; mutants = 18 12 & 7 embryos,,pubmed:39791977,,Whole body 45 hpf sox10 mutant embryos biol rep 2,GSM8655447,,source name:whole body|tissue:whole body|genotype:sox10 / |time:45 hpf|geo loc name:missing|collection date:missing,Whole body 45 hpf sox10 mutant embryos biol rep 2,The data was processed through the Galaxy project using Trimmomatic version 0.39 Sequences were pseudoaligned and quantified at TPM values using Kallisto. Assembly: GRCz11 Supplementary files format and content: Tab delimited text file includes TPM values for each transcript at 45 hpf 4 dpf and 7 dpf,whole body,,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina’s TruSeq® stranded mRNA library preparation kit.,,tissue:whole body|genotype:sox10 / |time:45 hpf,GSM8655447,GSM8655447: Whole body 45 hpf sox10 mutant embryos biol rep 2; Danio rerio; RNA Seq,GSM8655447 r1,GSM8655447,1,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina's TruSeq® stranded mRNA library preparation kit.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP548139,,,45 hpf_mut 2_R1.fastq.gz 45 hpf_mut 2_R2.fastq.gz,fastq fastq,15462167158.0,51199229.0,GSM8655447 r1,0:151 1:151,A:4089691508;C:3622289242;G:3495097207;T:4253826187;N:1263014,151,151,,,4089691508,3622289242,3495097207,4253826187,1263014,SRX26906742,SRS23386326,,,"Barske Lab, Human Genetics, Cincinnati Children's Hospital",,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2024-11-27,Multi-stage,Multi-stage,Trunk,Surface Structure 34220,SRR31539745,SRX26906741,SRS23386327,SRP548139,PRJNA1191617,Sox10 is required for systemic initiation of bone mineralization,GSE283071,Transcriptome Analysis,Heterozygous variants in SOX10 cause syndromes affecting pigmentation digestion hearing and neural development primarily attributable to failed differentiation or loss of non skeletal neural crest derivatives. We report here an additional novel requirement for Sox10 in bone mineralization. Neither crest nor mesoderm derived bones initiate mineralization on time in zebrafish sox10 mutants despite normal osteoblast differentiation and matrix production. Mutants are deficient in the Trpv6+ ionocytes that take up calcium from the environment resulting in severe calcium deficiency. As these ionocytes derive from ectoderm not crest we hypothesized that the primary defect resides in a separate organ that systemically regulates ionocyte numbers. RNAseq revealed significantly elevated stanniocalcin Stc1a an anti hypercalcemic hormone in sox10 mutants. Stc1a inhibits calcium uptake in fish by repressing trpv6 expression and Trpv6+ ionocyte proliferation. Epistasis assays confirm excess Stc1a as the proximate cause of the calcium deficit. The pronephros derived glands that synthesize Stc1a interact with sox10+ cells but these cells are missing in mutants. We conclude that sox10+ crest derived cells non autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary to initiate bone mineralization. Overall design: Bulk RNA seq was performed on pooled whole body zebrafish wild type and sox10 mutant larvae at 45 hpf 4 dpf and 7 dpf. Total of 18 samples. 45 hpf 3 replicates: controls n = 10 20 & 25; mutants n = 10 15 & 15 embryos 4 dpf 3 replicates: controls n = 10 15 & 12; mutants n = 10 11 & 12 embryos and 7 dpf 3 replicates: controls n = 12 30 & 18; mutants = 18 12 & 7 embryos,,pubmed:39791977,,Whole body 45 hpf sox10 mutant embryos biol rep 1,GSM8655446,,source name:whole body|tissue:whole body|genotype:sox10 / |time:45 hpf|geo loc name:missing|collection date:missing,Whole body 45 hpf sox10 mutant embryos biol rep 1,The data was processed through the Galaxy project using Trimmomatic version 0.39 Sequences were pseudoaligned and quantified at TPM values using Kallisto. Assembly: GRCz11 Supplementary files format and content: Tab delimited text file includes TPM values for each transcript at 45 hpf 4 dpf and 7 dpf,whole body,,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina’s TruSeq® stranded mRNA library preparation kit.,,tissue:whole body|genotype:sox10 / |time:45 hpf,GSM8655446,GSM8655446: Whole body 45 hpf sox10 mutant embryos biol rep 1; Danio rerio; RNA Seq,GSM8655446 r1,GSM8655446,1,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina's TruSeq® stranded mRNA library preparation kit.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP548139,,,45 hpf_mut 1_R1.fastq.gz 45 hpf_mut 1_R2.fastq.gz,fastq fastq,15655817712.0,51840456.0,GSM8655446 r1,0:151 1:151,A:4157360171;C:3674570903;G:3484564544;T:4338002005;N:1320089,151,151,,,4157360171,3674570903,3484564544,4338002005,1320089,SRX26906741,SRS23386327,,,"Barske Lab, Human Genetics, Cincinnati Children's Hospital",,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2024-11-27,Multi-stage,Multi-stage,Trunk,Surface Structure 34221,SRR31539746,SRX26906740,SRS23386328,SRP548139,PRJNA1191617,Sox10 is required for systemic initiation of bone mineralization,GSE283071,Transcriptome Analysis,Heterozygous variants in SOX10 cause syndromes affecting pigmentation digestion hearing and neural development primarily attributable to failed differentiation or loss of non skeletal neural crest derivatives. We report here an additional novel requirement for Sox10 in bone mineralization. Neither crest nor mesoderm derived bones initiate mineralization on time in zebrafish sox10 mutants despite normal osteoblast differentiation and matrix production. Mutants are deficient in the Trpv6+ ionocytes that take up calcium from the environment resulting in severe calcium deficiency. As these ionocytes derive from ectoderm not crest we hypothesized that the primary defect resides in a separate organ that systemically regulates ionocyte numbers. RNAseq revealed significantly elevated stanniocalcin Stc1a an anti hypercalcemic hormone in sox10 mutants. Stc1a inhibits calcium uptake in fish by repressing trpv6 expression and Trpv6+ ionocyte proliferation. Epistasis assays confirm excess Stc1a as the proximate cause of the calcium deficit. The pronephros derived glands that synthesize Stc1a interact with sox10+ cells but these cells are missing in mutants. We conclude that sox10+ crest derived cells non autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary to initiate bone mineralization. Overall design: Bulk RNA seq was performed on pooled whole body zebrafish wild type and sox10 mutant larvae at 45 hpf 4 dpf and 7 dpf. Total of 18 samples. 45 hpf 3 replicates: controls n = 10 20 & 25; mutants n = 10 15 & 15 embryos 4 dpf 3 replicates: controls n = 10 15 & 12; mutants n = 10 11 & 12 embryos and 7 dpf 3 replicates: controls n = 12 30 & 18; mutants = 18 12 & 7 embryos,,pubmed:39791977,,Whole body 45 hpf wild type embryos biol rep 3,GSM8655445,,source name:whole body|tissue:whole body|genotype:WT|time:45 hpf|geo loc name:missing|collection date:missing,Whole body 45 hpf wild type embryos biol rep 3,The data was processed through the Galaxy project using Trimmomatic version 0.39 Sequences were pseudoaligned and quantified at TPM values using Kallisto. Assembly: GRCz11 Supplementary files format and content: Tab delimited text file includes TPM values for each transcript at 45 hpf 4 dpf and 7 dpf,whole body,,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina’s TruSeq® stranded mRNA library preparation kit.,,tissue:whole body|genotype:WT|time:45 hpf,GSM8655445,GSM8655445: Whole body 45 hpf wild type embryos biol rep 3; Danio rerio; RNA Seq,GSM8655445 r1,GSM8655445,1,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina's TruSeq® stranded mRNA library preparation kit.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP548139,,,45 hpf_WT 3_R1.fastq.gz 45 hpf_WT 3_R2.fastq.gz,fastq fastq,14580346184.0,48279292.0,GSM8655445 r1,0:151 1:151,A:3858927638;C:3450290173;G:3271701209;T:3998202503;N:1224661,151,151,,,3858927638,3450290173,3271701209,3998202503,1224661,SRX26906740,SRS23386328,,,"Barske Lab, Human Genetics, Cincinnati Children's Hospital",,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2024-11-27,Multi-stage,Multi-stage,Trunk,Surface Structure 34222,SRR31539747,SRX26906739,SRS23386325,SRP548139,PRJNA1191617,Sox10 is required for systemic initiation of bone mineralization,GSE283071,Transcriptome Analysis,Heterozygous variants in SOX10 cause syndromes affecting pigmentation digestion hearing and neural development primarily attributable to failed differentiation or loss of non skeletal neural crest derivatives. We report here an additional novel requirement for Sox10 in bone mineralization. Neither crest nor mesoderm derived bones initiate mineralization on time in zebrafish sox10 mutants despite normal osteoblast differentiation and matrix production. Mutants are deficient in the Trpv6+ ionocytes that take up calcium from the environment resulting in severe calcium deficiency. As these ionocytes derive from ectoderm not crest we hypothesized that the primary defect resides in a separate organ that systemically regulates ionocyte numbers. RNAseq revealed significantly elevated stanniocalcin Stc1a an anti hypercalcemic hormone in sox10 mutants. Stc1a inhibits calcium uptake in fish by repressing trpv6 expression and Trpv6+ ionocyte proliferation. Epistasis assays confirm excess Stc1a as the proximate cause of the calcium deficit. The pronephros derived glands that synthesize Stc1a interact with sox10+ cells but these cells are missing in mutants. We conclude that sox10+ crest derived cells non autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary to initiate bone mineralization. Overall design: Bulk RNA seq was performed on pooled whole body zebrafish wild type and sox10 mutant larvae at 45 hpf 4 dpf and 7 dpf. Total of 18 samples. 45 hpf 3 replicates: controls n = 10 20 & 25; mutants n = 10 15 & 15 embryos 4 dpf 3 replicates: controls n = 10 15 & 12; mutants n = 10 11 & 12 embryos and 7 dpf 3 replicates: controls n = 12 30 & 18; mutants = 18 12 & 7 embryos,,pubmed:39791977,,Whole body 45 hpf wild type embryos biol rep 2,GSM8655444,,source name:whole body|tissue:whole body|genotype:WT|time:45 hpf|geo loc name:missing|collection date:missing,Whole body 45 hpf wild type embryos biol rep 2,The data was processed through the Galaxy project using Trimmomatic version 0.39 Sequences were pseudoaligned and quantified at TPM values using Kallisto. Assembly: GRCz11 Supplementary files format and content: Tab delimited text file includes TPM values for each transcript at 45 hpf 4 dpf and 7 dpf,whole body,,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina’s TruSeq® stranded mRNA library preparation kit.,,tissue:whole body|genotype:WT|time:45 hpf,GSM8655444,GSM8655444: Whole body 45 hpf wild type embryos biol rep 2; Danio rerio; RNA Seq,GSM8655444 r1,GSM8655444,1,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina's TruSeq® stranded mRNA library preparation kit.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP548139,,,45 hpf_WT 2_R1.fastq.gz 45 hpf_WT_2_R2.fastq.gz,fastq fastq,15005198878.0,49686089.0,GSM8655444 r1,0:151 1:151,A:3950487195;C:3509517845;G:3418800225;T:4125159820;N:1233793,151,151,,,3950487195,3509517845,3418800225,4125159820,1233793,SRX26906739,SRS23386325,,,"Barske Lab, Human Genetics, Cincinnati Children's Hospital",,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2024-11-27,Multi-stage,Multi-stage,Trunk,Surface Structure 34223,SRR31539748,SRX26906738,SRS23386324,SRP548139,PRJNA1191617,Sox10 is required for systemic initiation of bone mineralization,GSE283071,Transcriptome Analysis,Heterozygous variants in SOX10 cause syndromes affecting pigmentation digestion hearing and neural development primarily attributable to failed differentiation or loss of non skeletal neural crest derivatives. We report here an additional novel requirement for Sox10 in bone mineralization. Neither crest nor mesoderm derived bones initiate mineralization on time in zebrafish sox10 mutants despite normal osteoblast differentiation and matrix production. Mutants are deficient in the Trpv6+ ionocytes that take up calcium from the environment resulting in severe calcium deficiency. As these ionocytes derive from ectoderm not crest we hypothesized that the primary defect resides in a separate organ that systemically regulates ionocyte numbers. RNAseq revealed significantly elevated stanniocalcin Stc1a an anti hypercalcemic hormone in sox10 mutants. Stc1a inhibits calcium uptake in fish by repressing trpv6 expression and Trpv6+ ionocyte proliferation. Epistasis assays confirm excess Stc1a as the proximate cause of the calcium deficit. The pronephros derived glands that synthesize Stc1a interact with sox10+ cells but these cells are missing in mutants. We conclude that sox10+ crest derived cells non autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary to initiate bone mineralization. Overall design: Bulk RNA seq was performed on pooled whole body zebrafish wild type and sox10 mutant larvae at 45 hpf 4 dpf and 7 dpf. Total of 18 samples. 45 hpf 3 replicates: controls n = 10 20 & 25; mutants n = 10 15 & 15 embryos 4 dpf 3 replicates: controls n = 10 15 & 12; mutants n = 10 11 & 12 embryos and 7 dpf 3 replicates: controls n = 12 30 & 18; mutants = 18 12 & 7 embryos,,pubmed:39791977,,Whole body 45 hpf wild type embryos biol rep 1,GSM8655443,,source name:whole body|tissue:whole body|genotype:WT|time:45 hpf|geo loc name:missing|collection date:missing,Whole body 45 hpf wild type embryos biol rep 1,The data was processed through the Galaxy project using Trimmomatic version 0.39 Sequences were pseudoaligned and quantified at TPM values using Kallisto. Assembly: GRCz11 Supplementary files format and content: Tab delimited text file includes TPM values for each transcript at 45 hpf 4 dpf and 7 dpf,whole body,,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina’s TruSeq® stranded mRNA library preparation kit.,,tissue:whole body|genotype:WT|time:45 hpf,GSM8655443,GSM8655443: Whole body 45 hpf wild type embryos biol rep 1; Danio rerio; RNA Seq,GSM8655443 r1,GSM8655443,1,RNA was extracted using the RNAqueous 4PCR Total RNA Isolation Kit Invitrogen 150 to 300 ng of total RNA determined by InvitrogenTM QubitTM high sensitivity spectrofluorometric measurement was poly A selected and reverse transcribed using Illumina's TruSeq® stranded mRNA library preparation kit.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP548139,,,45 hpf_WT 1_R1.fastq.gz 45 hpf_WT 1_R2.fastq.gz,fastq fastq,14884992912.0,49288056.0,GSM8655443 r1,0:151 1:151,A:3878762924;C:3475070121;G:3485186845;T:4044768200;N:1204822,151,151,,,3878762924,3475070121,3485186845,4044768200,1204822,SRX26906738,SRS23386324,,,"Barske Lab, Human Genetics, Cincinnati Children's Hospital",,,,,,,,,,,,B,B,biological fallback assumption,illumina,novaseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2024-11-27,Multi-stage,Multi-stage,Trunk,Surface Structure 35871,SRR33167498,SRX28428354,SRS24751227,SRP579141,PRJNA1251588,Investigation into molecular mechanisms behind microbiome and normal neurobehavioral development in zebrafish,GSE294778,Transcriptome Analysis,Changes in resident microbiota may have wide ranging effects on human health. Previous research found axenic zebrafish Danio rerio exhibit hyperactivity at 10 dpf dpf. This abnormal neurobehavioral response can be prevented with re colonization of axenic embryos with microbes at 1 dpf. The present study investigated the molecular mechanism behind this effect through RNA sequencing of pooled 10 dpf larval head tissue from axenic conventionally colonized controls and axenic zebrafish recolonized with microbes at 1 dpf. Overall design: 10 dpf dpf axenic microbe free conventionally colonized normal and axenic Danio rerio embryos recolonized with microbes at 1 dpf control for axenic derivation process were submitted for RNA sequencing to investigate the molecular mechanism behind hyperactivity in axenic larvae.,,,,MB125 2B L6.D708,GSM8919410,,source name:pooled heads collected anterior to swim bladder of larval zebrafish|tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:1|geo loc name:missing|collection date:missing,MB125 2B L6.D708,For Experiment 1 basecall files were converted to FASTQ by bcl2fastq2 demultiplexed quality trimmed and filter using ea utils https://expressionanalysis.github.io/ea utils/ ; last accessed 01 21 2020. Reads with Phred quality scores <7 or >4 Ns at either end of the read were trimmed. Reads with any one base comprising > 95% frequency of the read or having an average Phred quality score <25 were removed. For Experiment 2 Illumina basecall files were converted to FASTQ and demultiplexed by bcl2fastq2. Reads with 3’ Phred quality scores < 20 or lengths <25 nucleotides were trimmed or removed respectively in Partek Flow v.8.0.19.0428; St. Louis MO. Experiments 1 and 2. Trimmed FASTQ files from both sequencing experiments were aligned to Danio rerio GRCz11 using STAR v.2.5.3a with default settings in Partek Flow and strand specificity detection enabled for the paired end reads from Experiment 1 but not for Experiment 2. Gene counts from both experiments were quantified from aligned reads using Partek Expectation Maximization and Ensembl Danio rerio GRCz11.93 gene annotation. Assembly: GRCz11/93 gene annotation Supplementary files format and content: tab delimited file for experiment1 containing raw counts for all samples Supplementary files format and content: tab delimited file for experiment2 containing raw counts for all samples,pooled heads collected anterior to swim bladder of larval zebrafish,,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product’s quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:1,GSM8919410,GSM8919410: MB125 2B L6.D708; Danio rerio; RNA Seq,GSM8919410 r1,GSM8919410,1,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product's quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP579141,,,MB125_2B_L6.D708_1.fastq.gz MB125_2B_L6.D708_2.fastq.gz,fastq fastq,2319886164.0,22743982.0,GSM8919410 r1,0:51 1:51,A:553297266;C:587452449;G:584241025;T:594875512;N:19912,51,51,,,553297266,587452449,584241025,594875512,19912,SRX28428354,SRS24751227,SRA2113523,US EPA,US EPA,,,,,,,,,,,,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2025-04-16,Multi-stage,Multi-stage,Multi-tissue,Multi-system 35892,SRR33167519,SRX28428333,SRS24751206,SRP579141,PRJNA1251588,Investigation into molecular mechanisms behind microbiome and normal neurobehavioral development in zebrafish,GSE294778,Transcriptome Analysis,Changes in resident microbiota may have wide ranging effects on human health. Previous research found axenic zebrafish Danio rerio exhibit hyperactivity at 10 dpf dpf. This abnormal neurobehavioral response can be prevented with re colonization of axenic embryos with microbes at 1 dpf. The present study investigated the molecular mechanism behind this effect through RNA sequencing of pooled 10 dpf larval head tissue from axenic conventionally colonized controls and axenic zebrafish recolonized with microbes at 1 dpf. Overall design: 10 dpf dpf axenic microbe free conventionally colonized normal and axenic Danio rerio embryos recolonized with microbes at 1 dpf control for axenic derivation process were submitted for RNA sequencing to investigate the molecular mechanism behind hyperactivity in axenic larvae.,,,,GRC355 AC7 S7,GSM8919392,,source name:pooled heads collected anterior to swim bladder of larval zebrafish|tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:2|geo loc name:missing|collection date:missing,GRC355 AC7 S7,For Experiment 1 basecall files were converted to FASTQ by bcl2fastq2 demultiplexed quality trimmed and filter using ea utils https://expressionanalysis.github.io/ea utils/ ; last accessed 01 21 2020. Reads with Phred quality scores <7 or >4 Ns at either end of the read were trimmed. Reads with any one base comprising > 95% frequency of the read or having an average Phred quality score <25 were removed. For Experiment 2 Illumina basecall files were converted to FASTQ and demultiplexed by bcl2fastq2. Reads with 3’ Phred quality scores < 20 or lengths <25 nucleotides were trimmed or removed respectively in Partek Flow v.8.0.19.0428; St. Louis MO. Experiments 1 and 2. Trimmed FASTQ files from both sequencing experiments were aligned to Danio rerio GRCz11 using STAR v.2.5.3a with default settings in Partek Flow and strand specificity detection enabled for the paired end reads from Experiment 1 but not for Experiment 2. Gene counts from both experiments were quantified from aligned reads using Partek Expectation Maximization and Ensembl Danio rerio GRCz11.93 gene annotation. Assembly: GRCz11/93 gene annotation Supplementary files format and content: tab delimited file for experiment1 containing raw counts for all samples Supplementary files format and content: tab delimited file for experiment2 containing raw counts for all samples,pooled heads collected anterior to swim bladder of larval zebrafish,,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product’s quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:2,GSM8919392,GSM8919392: GRC355 AC7 S7; Danio rerio; RNA Seq,GSM8919392 r1,GSM8919392,1,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product's quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP579141,,,GRC355_AC7_S7_R1_001.fastq.gz,fastq,2227335150.0,29697802.0,GSM8919392 r1,0:75,A:600851630;C:506204586;G:559397063;T:560849750;N:32121,75,,,,600851630,506204586,559397063,560849750,32121,SRX28428333,SRS24751206,SRA2113523,US EPA,US EPA,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2025-04-16,Multi-stage,Multi-stage,Multi-tissue,Multi-system 35893,SRR33167520,SRX28428332,SRS24751204,SRP579141,PRJNA1251588,Investigation into molecular mechanisms behind microbiome and normal neurobehavioral development in zebrafish,GSE294778,Transcriptome Analysis,Changes in resident microbiota may have wide ranging effects on human health. Previous research found axenic zebrafish Danio rerio exhibit hyperactivity at 10 dpf dpf. This abnormal neurobehavioral response can be prevented with re colonization of axenic embryos with microbes at 1 dpf. The present study investigated the molecular mechanism behind this effect through RNA sequencing of pooled 10 dpf larval head tissue from axenic conventionally colonized controls and axenic zebrafish recolonized with microbes at 1 dpf. Overall design: 10 dpf dpf axenic microbe free conventionally colonized normal and axenic Danio rerio embryos recolonized with microbes at 1 dpf control for axenic derivation process were submitted for RNA sequencing to investigate the molecular mechanism behind hyperactivity in axenic larvae.,,,,GRC355 AC6 S11,GSM8919391,,source name:pooled heads collected anterior to swim bladder of larval zebrafish|tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:2|geo loc name:missing|collection date:missing,GRC355 AC6 S11,For Experiment 1 basecall files were converted to FASTQ by bcl2fastq2 demultiplexed quality trimmed and filter using ea utils https://expressionanalysis.github.io/ea utils/ ; last accessed 01 21 2020. Reads with Phred quality scores <7 or >4 Ns at either end of the read were trimmed. Reads with any one base comprising > 95% frequency of the read or having an average Phred quality score <25 were removed. For Experiment 2 Illumina basecall files were converted to FASTQ and demultiplexed by bcl2fastq2. Reads with 3’ Phred quality scores < 20 or lengths <25 nucleotides were trimmed or removed respectively in Partek Flow v.8.0.19.0428; St. Louis MO. Experiments 1 and 2. Trimmed FASTQ files from both sequencing experiments were aligned to Danio rerio GRCz11 using STAR v.2.5.3a with default settings in Partek Flow and strand specificity detection enabled for the paired end reads from Experiment 1 but not for Experiment 2. Gene counts from both experiments were quantified from aligned reads using Partek Expectation Maximization and Ensembl Danio rerio GRCz11.93 gene annotation. Assembly: GRCz11/93 gene annotation Supplementary files format and content: tab delimited file for experiment1 containing raw counts for all samples Supplementary files format and content: tab delimited file for experiment2 containing raw counts for all samples,pooled heads collected anterior to swim bladder of larval zebrafish,,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product’s quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:2,GSM8919391,GSM8919391: GRC355 AC6 S11; Danio rerio; RNA Seq,GSM8919391 r1,GSM8919391,1,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product's quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP579141,,,GRC355_AC6_S11_R1_001.fastq.gz,fastq,2585681925.0,34475759.0,GSM8919391 r1,0:75,A:689801171;C:608098568;G:666027069;T:621732490;N:22627,75,,,,689801171,608098568,666027069,621732490,22627,SRX28428332,SRS24751204,SRA2113523,US EPA,US EPA,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2025-04-16,Multi-stage,Multi-stage,Multi-tissue,Multi-system 35894,SRR33167521,SRX28428331,SRS24751205,SRP579141,PRJNA1251588,Investigation into molecular mechanisms behind microbiome and normal neurobehavioral development in zebrafish,GSE294778,Transcriptome Analysis,Changes in resident microbiota may have wide ranging effects on human health. Previous research found axenic zebrafish Danio rerio exhibit hyperactivity at 10 dpf dpf. This abnormal neurobehavioral response can be prevented with re colonization of axenic embryos with microbes at 1 dpf. The present study investigated the molecular mechanism behind this effect through RNA sequencing of pooled 10 dpf larval head tissue from axenic conventionally colonized controls and axenic zebrafish recolonized with microbes at 1 dpf. Overall design: 10 dpf dpf axenic microbe free conventionally colonized normal and axenic Danio rerio embryos recolonized with microbes at 1 dpf control for axenic derivation process were submitted for RNA sequencing to investigate the molecular mechanism behind hyperactivity in axenic larvae.,,,,GRC355 AC5 S14,GSM8919390,,source name:pooled heads collected anterior to swim bladder of larval zebrafish|tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:2|geo loc name:missing|collection date:missing,GRC355 AC5 S14,For Experiment 1 basecall files were converted to FASTQ by bcl2fastq2 demultiplexed quality trimmed and filter using ea utils https://expressionanalysis.github.io/ea utils/ ; last accessed 01 21 2020. Reads with Phred quality scores <7 or >4 Ns at either end of the read were trimmed. Reads with any one base comprising > 95% frequency of the read or having an average Phred quality score <25 were removed. For Experiment 2 Illumina basecall files were converted to FASTQ and demultiplexed by bcl2fastq2. Reads with 3’ Phred quality scores < 20 or lengths <25 nucleotides were trimmed or removed respectively in Partek Flow v.8.0.19.0428; St. Louis MO. Experiments 1 and 2. Trimmed FASTQ files from both sequencing experiments were aligned to Danio rerio GRCz11 using STAR v.2.5.3a with default settings in Partek Flow and strand specificity detection enabled for the paired end reads from Experiment 1 but not for Experiment 2. Gene counts from both experiments were quantified from aligned reads using Partek Expectation Maximization and Ensembl Danio rerio GRCz11.93 gene annotation. Assembly: GRCz11/93 gene annotation Supplementary files format and content: tab delimited file for experiment1 containing raw counts for all samples Supplementary files format and content: tab delimited file for experiment2 containing raw counts for all samples,pooled heads collected anterior to swim bladder of larval zebrafish,,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product’s quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:2,GSM8919390,GSM8919390: GRC355 AC5 S14; Danio rerio; RNA Seq,GSM8919390 r1,GSM8919390,1,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product's quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP579141,,,GRC355_AC5_S14_R1_001.fastq.gz,fastq,2494451250.0,33259350.0,GSM8919390 r1,0:75,A:663836714;C:570411156;G:619148577;T:641019188;N:35615,75,,,,663836714,570411156,619148577,641019188,35615,SRX28428331,SRS24751205,SRA2113523,US EPA,US EPA,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2025-04-16,Multi-stage,Multi-stage,Multi-tissue,Multi-system 35895,SRR33167522,SRX28428330,SRS24751203,SRP579141,PRJNA1251588,Investigation into molecular mechanisms behind microbiome and normal neurobehavioral development in zebrafish,GSE294778,Transcriptome Analysis,Changes in resident microbiota may have wide ranging effects on human health. Previous research found axenic zebrafish Danio rerio exhibit hyperactivity at 10 dpf dpf. This abnormal neurobehavioral response can be prevented with re colonization of axenic embryos with microbes at 1 dpf. The present study investigated the molecular mechanism behind this effect through RNA sequencing of pooled 10 dpf larval head tissue from axenic conventionally colonized controls and axenic zebrafish recolonized with microbes at 1 dpf. Overall design: 10 dpf dpf axenic microbe free conventionally colonized normal and axenic Danio rerio embryos recolonized with microbes at 1 dpf control for axenic derivation process were submitted for RNA sequencing to investigate the molecular mechanism behind hyperactivity in axenic larvae.,,,,GRC355 AC4 S16,GSM8919389,,source name:pooled heads collected anterior to swim bladder of larval zebrafish|tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:2|geo loc name:missing|collection date:missing,GRC355 AC4 S16,For Experiment 1 basecall files were converted to FASTQ by bcl2fastq2 demultiplexed quality trimmed and filter using ea utils https://expressionanalysis.github.io/ea utils/ ; last accessed 01 21 2020. Reads with Phred quality scores <7 or >4 Ns at either end of the read were trimmed. Reads with any one base comprising > 95% frequency of the read or having an average Phred quality score <25 were removed. For Experiment 2 Illumina basecall files were converted to FASTQ and demultiplexed by bcl2fastq2. Reads with 3’ Phred quality scores < 20 or lengths <25 nucleotides were trimmed or removed respectively in Partek Flow v.8.0.19.0428; St. Louis MO. Experiments 1 and 2. Trimmed FASTQ files from both sequencing experiments were aligned to Danio rerio GRCz11 using STAR v.2.5.3a with default settings in Partek Flow and strand specificity detection enabled for the paired end reads from Experiment 1 but not for Experiment 2. Gene counts from both experiments were quantified from aligned reads using Partek Expectation Maximization and Ensembl Danio rerio GRCz11.93 gene annotation. Assembly: GRCz11/93 gene annotation Supplementary files format and content: tab delimited file for experiment1 containing raw counts for all samples Supplementary files format and content: tab delimited file for experiment2 containing raw counts for all samples,pooled heads collected anterior to swim bladder of larval zebrafish,,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product’s quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:2,GSM8919389,GSM8919389: GRC355 AC4 S16; Danio rerio; RNA Seq,GSM8919389 r1,GSM8919389,1,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product's quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP579141,,,GRC355_AC4_S16_R1_001.fastq.gz,fastq,2318944500.0,30919260.0,GSM8919389 r1,0:75,A:622730628;C:558676778;G:600421168;T:537095873;N:20053,75,,,,622730628,558676778,600421168,537095873,20053,SRX28428330,SRS24751203,SRA2113523,US EPA,US EPA,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2025-04-16,Multi-stage,Multi-stage,Multi-tissue,Multi-system 35896,SRR33167523,SRX28428329,SRS24751201,SRP579141,PRJNA1251588,Investigation into molecular mechanisms behind microbiome and normal neurobehavioral development in zebrafish,GSE294778,Transcriptome Analysis,Changes in resident microbiota may have wide ranging effects on human health. Previous research found axenic zebrafish Danio rerio exhibit hyperactivity at 10 dpf dpf. This abnormal neurobehavioral response can be prevented with re colonization of axenic embryos with microbes at 1 dpf. The present study investigated the molecular mechanism behind this effect through RNA sequencing of pooled 10 dpf larval head tissue from axenic conventionally colonized controls and axenic zebrafish recolonized with microbes at 1 dpf. Overall design: 10 dpf dpf axenic microbe free conventionally colonized normal and axenic Danio rerio embryos recolonized with microbes at 1 dpf control for axenic derivation process were submitted for RNA sequencing to investigate the molecular mechanism behind hyperactivity in axenic larvae.,,,,GRC355 AC3 S10,GSM8919388,,source name:pooled heads collected anterior to swim bladder of larval zebrafish|tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:2|geo loc name:missing|collection date:missing,GRC355 AC3 S10,For Experiment 1 basecall files were converted to FASTQ by bcl2fastq2 demultiplexed quality trimmed and filter using ea utils https://expressionanalysis.github.io/ea utils/ ; last accessed 01 21 2020. Reads with Phred quality scores <7 or >4 Ns at either end of the read were trimmed. Reads with any one base comprising > 95% frequency of the read or having an average Phred quality score <25 were removed. For Experiment 2 Illumina basecall files were converted to FASTQ and demultiplexed by bcl2fastq2. Reads with 3’ Phred quality scores < 20 or lengths <25 nucleotides were trimmed or removed respectively in Partek Flow v.8.0.19.0428; St. Louis MO. Experiments 1 and 2. Trimmed FASTQ files from both sequencing experiments were aligned to Danio rerio GRCz11 using STAR v.2.5.3a with default settings in Partek Flow and strand specificity detection enabled for the paired end reads from Experiment 1 but not for Experiment 2. Gene counts from both experiments were quantified from aligned reads using Partek Expectation Maximization and Ensembl Danio rerio GRCz11.93 gene annotation. Assembly: GRCz11/93 gene annotation Supplementary files format and content: tab delimited file for experiment1 containing raw counts for all samples Supplementary files format and content: tab delimited file for experiment2 containing raw counts for all samples,pooled heads collected anterior to swim bladder of larval zebrafish,,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product’s quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:2,GSM8919388,GSM8919388: GRC355 AC3 S10; Danio rerio; RNA Seq,GSM8919388 r1,GSM8919388,1,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product's quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP579141,,,GRC355_AC3_S10_R1_001.fastq.gz,fastq,2294756475.0,30596753.0,GSM8919388 r1,0:75,A:602530943;C:536584842;G:585162453;T:570445286;N:32951,75,,,,602530943,536584842,585162453,570445286,32951,SRX28428329,SRS24751201,SRA2113523,US EPA,US EPA,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2025-04-16,Multi-stage,Multi-stage,Multi-tissue,Multi-system 35897,SRR33167524,SRX28428328,SRS24751202,SRP579141,PRJNA1251588,Investigation into molecular mechanisms behind microbiome and normal neurobehavioral development in zebrafish,GSE294778,Transcriptome Analysis,Changes in resident microbiota may have wide ranging effects on human health. Previous research found axenic zebrafish Danio rerio exhibit hyperactivity at 10 dpf dpf. This abnormal neurobehavioral response can be prevented with re colonization of axenic embryos with microbes at 1 dpf. The present study investigated the molecular mechanism behind this effect through RNA sequencing of pooled 10 dpf larval head tissue from axenic conventionally colonized controls and axenic zebrafish recolonized with microbes at 1 dpf. Overall design: 10 dpf dpf axenic microbe free conventionally colonized normal and axenic Danio rerio embryos recolonized with microbes at 1 dpf control for axenic derivation process were submitted for RNA sequencing to investigate the molecular mechanism behind hyperactivity in axenic larvae.,,,,GRC355 AC2 S1,GSM8919387,,source name:pooled heads collected anterior to swim bladder of larval zebrafish|tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:2|geo loc name:missing|collection date:missing,GRC355 AC2 S1,For Experiment 1 basecall files were converted to FASTQ by bcl2fastq2 demultiplexed quality trimmed and filter using ea utils https://expressionanalysis.github.io/ea utils/ ; last accessed 01 21 2020. Reads with Phred quality scores <7 or >4 Ns at either end of the read were trimmed. Reads with any one base comprising > 95% frequency of the read or having an average Phred quality score <25 were removed. For Experiment 2 Illumina basecall files were converted to FASTQ and demultiplexed by bcl2fastq2. Reads with 3’ Phred quality scores < 20 or lengths <25 nucleotides were trimmed or removed respectively in Partek Flow v.8.0.19.0428; St. Louis MO. Experiments 1 and 2. Trimmed FASTQ files from both sequencing experiments were aligned to Danio rerio GRCz11 using STAR v.2.5.3a with default settings in Partek Flow and strand specificity detection enabled for the paired end reads from Experiment 1 but not for Experiment 2. Gene counts from both experiments were quantified from aligned reads using Partek Expectation Maximization and Ensembl Danio rerio GRCz11.93 gene annotation. Assembly: GRCz11/93 gene annotation Supplementary files format and content: tab delimited file for experiment1 containing raw counts for all samples Supplementary files format and content: tab delimited file for experiment2 containing raw counts for all samples,pooled heads collected anterior to swim bladder of larval zebrafish,,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product’s quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:2,GSM8919387,GSM8919387: GRC355 AC2 S1; Danio rerio; RNA Seq,GSM8919387 r1,GSM8919387,1,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product's quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP579141,,,GRC355_AC2_S1_R1_001.fastq.gz,fastq,2339756250.0,31196750.0,GSM8919387 r1,0:75,A:612473749;C:554816637;G:611772519;T:560660557;N:32788,75,,,,612473749,554816637,611772519,560660557,32788,SRX28428328,SRS24751202,SRA2113523,US EPA,US EPA,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2025-04-16,Multi-stage,Multi-stage,Multi-tissue,Multi-system 35901,SRR33167528,SRX28428324,SRS24751197,SRP579141,PRJNA1251588,Investigation into molecular mechanisms behind microbiome and normal neurobehavioral development in zebrafish,GSE294778,Transcriptome Analysis,Changes in resident microbiota may have wide ranging effects on human health. Previous research found axenic zebrafish Danio rerio exhibit hyperactivity at 10 dpf dpf. This abnormal neurobehavioral response can be prevented with re colonization of axenic embryos with microbes at 1 dpf. The present study investigated the molecular mechanism behind this effect through RNA sequencing of pooled 10 dpf larval head tissue from axenic conventionally colonized controls and axenic zebrafish recolonized with microbes at 1 dpf. Overall design: 10 dpf dpf axenic microbe free conventionally colonized normal and axenic Danio rerio embryos recolonized with microbes at 1 dpf control for axenic derivation process were submitted for RNA sequencing to investigate the molecular mechanism behind hyperactivity in axenic larvae.,,,,MB125 2H L6.D701,GSM8919415,,source name:pooled heads collected anterior to swim bladder of larval zebrafish|tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:1|geo loc name:missing|collection date:missing,MB125 2H L6.D701,For Experiment 1 basecall files were converted to FASTQ by bcl2fastq2 demultiplexed quality trimmed and filter using ea utils https://expressionanalysis.github.io/ea utils/ ; last accessed 01 21 2020. Reads with Phred quality scores <7 or >4 Ns at either end of the read were trimmed. Reads with any one base comprising > 95% frequency of the read or having an average Phred quality score <25 were removed. For Experiment 2 Illumina basecall files were converted to FASTQ and demultiplexed by bcl2fastq2. Reads with 3’ Phred quality scores < 20 or lengths <25 nucleotides were trimmed or removed respectively in Partek Flow v.8.0.19.0428; St. Louis MO. Experiments 1 and 2. Trimmed FASTQ files from both sequencing experiments were aligned to Danio rerio GRCz11 using STAR v.2.5.3a with default settings in Partek Flow and strand specificity detection enabled for the paired end reads from Experiment 1 but not for Experiment 2. Gene counts from both experiments were quantified from aligned reads using Partek Expectation Maximization and Ensembl Danio rerio GRCz11.93 gene annotation. Assembly: GRCz11/93 gene annotation Supplementary files format and content: tab delimited file for experiment1 containing raw counts for all samples Supplementary files format and content: tab delimited file for experiment2 containing raw counts for all samples,pooled heads collected anterior to swim bladder of larval zebrafish,,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product’s quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:1,GSM8919415,GSM8919415: MB125 2H L6.D701; Danio rerio; RNA Seq,GSM8919415 r1,GSM8919415,1,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product's quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP579141,,,MB125_2H_L6.D701_1.fastq.gz MB125_2H_L6.D701_2.fastq.gz,fastq fastq,2248766358.0,22046729.0,GSM8919415 r1,0:51 1:51,A:535472034;C:572268414;G:569258495;T:571751143;N:16272,51,51,,,535472034,572268414,569258495,571751143,16272,SRX28428324,SRS24751197,SRA2113523,US EPA,US EPA,,,,,,,,,,,,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2025-04-16,Multi-stage,Multi-stage,Multi-tissue,Multi-system 35902,SRR33167529,SRX28428323,SRS24751196,SRP579141,PRJNA1251588,Investigation into molecular mechanisms behind microbiome and normal neurobehavioral development in zebrafish,GSE294778,Transcriptome Analysis,Changes in resident microbiota may have wide ranging effects on human health. Previous research found axenic zebrafish Danio rerio exhibit hyperactivity at 10 dpf dpf. This abnormal neurobehavioral response can be prevented with re colonization of axenic embryos with microbes at 1 dpf. The present study investigated the molecular mechanism behind this effect through RNA sequencing of pooled 10 dpf larval head tissue from axenic conventionally colonized controls and axenic zebrafish recolonized with microbes at 1 dpf. Overall design: 10 dpf dpf axenic microbe free conventionally colonized normal and axenic Danio rerio embryos recolonized with microbes at 1 dpf control for axenic derivation process were submitted for RNA sequencing to investigate the molecular mechanism behind hyperactivity in axenic larvae.,,,,MB125 2G L6.D711,GSM8919414,,source name:pooled heads collected anterior to swim bladder of larval zebrafish|tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:1|geo loc name:missing|collection date:missing,MB125 2G L6.D711,For Experiment 1 basecall files were converted to FASTQ by bcl2fastq2 demultiplexed quality trimmed and filter using ea utils https://expressionanalysis.github.io/ea utils/ ; last accessed 01 21 2020. Reads with Phred quality scores <7 or >4 Ns at either end of the read were trimmed. Reads with any one base comprising > 95% frequency of the read or having an average Phred quality score <25 were removed. For Experiment 2 Illumina basecall files were converted to FASTQ and demultiplexed by bcl2fastq2. Reads with 3’ Phred quality scores < 20 or lengths <25 nucleotides were trimmed or removed respectively in Partek Flow v.8.0.19.0428; St. Louis MO. Experiments 1 and 2. Trimmed FASTQ files from both sequencing experiments were aligned to Danio rerio GRCz11 using STAR v.2.5.3a with default settings in Partek Flow and strand specificity detection enabled for the paired end reads from Experiment 1 but not for Experiment 2. Gene counts from both experiments were quantified from aligned reads using Partek Expectation Maximization and Ensembl Danio rerio GRCz11.93 gene annotation. Assembly: GRCz11/93 gene annotation Supplementary files format and content: tab delimited file for experiment1 containing raw counts for all samples Supplementary files format and content: tab delimited file for experiment2 containing raw counts for all samples,pooled heads collected anterior to swim bladder of larval zebrafish,,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product’s quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:1,GSM8919414,GSM8919414: MB125 2G L6.D711; Danio rerio; RNA Seq,GSM8919414 r1,GSM8919414,1,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product's quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP579141,,,MB125_2G_L6.D711_1.fastq.gz MB125_2G_L6.D711_2.fastq.gz,fastq fastq,2198049510.0,21549505.0,GSM8919414 r1,0:51 1:51,A:535878049;C:547290891;G:544359481;T:570502976;N:18113,51,51,,,535878049,547290891,544359481,570502976,18113,SRX28428323,SRS24751196,SRA2113523,US EPA,US EPA,,,,,,,,,,,,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2025-04-16,Multi-stage,Multi-stage,Multi-tissue,Multi-system 35903,SRR33167530,SRX28428322,SRS24751195,SRP579141,PRJNA1251588,Investigation into molecular mechanisms behind microbiome and normal neurobehavioral development in zebrafish,GSE294778,Transcriptome Analysis,Changes in resident microbiota may have wide ranging effects on human health. Previous research found axenic zebrafish Danio rerio exhibit hyperactivity at 10 dpf dpf. This abnormal neurobehavioral response can be prevented with re colonization of axenic embryos with microbes at 1 dpf. The present study investigated the molecular mechanism behind this effect through RNA sequencing of pooled 10 dpf larval head tissue from axenic conventionally colonized controls and axenic zebrafish recolonized with microbes at 1 dpf. Overall design: 10 dpf dpf axenic microbe free conventionally colonized normal and axenic Danio rerio embryos recolonized with microbes at 1 dpf control for axenic derivation process were submitted for RNA sequencing to investigate the molecular mechanism behind hyperactivity in axenic larvae.,,,,MB125 2F L6.D712,GSM8919413,,source name:pooled heads collected anterior to swim bladder of larval zebrafish|tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:1|geo loc name:missing|collection date:missing,MB125 2F L6.D712,For Experiment 1 basecall files were converted to FASTQ by bcl2fastq2 demultiplexed quality trimmed and filter using ea utils https://expressionanalysis.github.io/ea utils/ ; last accessed 01 21 2020. Reads with Phred quality scores <7 or >4 Ns at either end of the read were trimmed. Reads with any one base comprising > 95% frequency of the read or having an average Phred quality score <25 were removed. For Experiment 2 Illumina basecall files were converted to FASTQ and demultiplexed by bcl2fastq2. Reads with 3’ Phred quality scores < 20 or lengths <25 nucleotides were trimmed or removed respectively in Partek Flow v.8.0.19.0428; St. Louis MO. Experiments 1 and 2. Trimmed FASTQ files from both sequencing experiments were aligned to Danio rerio GRCz11 using STAR v.2.5.3a with default settings in Partek Flow and strand specificity detection enabled for the paired end reads from Experiment 1 but not for Experiment 2. Gene counts from both experiments were quantified from aligned reads using Partek Expectation Maximization and Ensembl Danio rerio GRCz11.93 gene annotation. Assembly: GRCz11/93 gene annotation Supplementary files format and content: tab delimited file for experiment1 containing raw counts for all samples Supplementary files format and content: tab delimited file for experiment2 containing raw counts for all samples,pooled heads collected anterior to swim bladder of larval zebrafish,,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product’s quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:1,GSM8919413,GSM8919413: MB125 2F L6.D712; Danio rerio; RNA Seq,GSM8919413 r1,GSM8919413,1,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product's quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP579141,,,MB125_2F_L6.D712_1.fastq.gz MB125_2F_L6.D712_2.fastq.gz,fastq fastq,2601840378.0,25508239.0,GSM8919413 r1,0:51 1:51,A:584354104;C:693159013;G:696042575;T:628262140;N:22546,51,51,,,584354104,693159013,696042575,628262140,22546,SRX28428322,SRS24751195,SRA2113523,US EPA,US EPA,,,,,,,,,,,,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2025-04-16,Multi-stage,Multi-stage,Multi-tissue,Multi-system 35904,SRR33167531,SRX28428321,SRS24751194,SRP579141,PRJNA1251588,Investigation into molecular mechanisms behind microbiome and normal neurobehavioral development in zebrafish,GSE294778,Transcriptome Analysis,Changes in resident microbiota may have wide ranging effects on human health. Previous research found axenic zebrafish Danio rerio exhibit hyperactivity at 10 dpf dpf. This abnormal neurobehavioral response can be prevented with re colonization of axenic embryos with microbes at 1 dpf. The present study investigated the molecular mechanism behind this effect through RNA sequencing of pooled 10 dpf larval head tissue from axenic conventionally colonized controls and axenic zebrafish recolonized with microbes at 1 dpf. Overall design: 10 dpf dpf axenic microbe free conventionally colonized normal and axenic Danio rerio embryos recolonized with microbes at 1 dpf control for axenic derivation process were submitted for RNA sequencing to investigate the molecular mechanism behind hyperactivity in axenic larvae.,,,,MB125 2E L6.D709,GSM8919412,,source name:pooled heads collected anterior to swim bladder of larval zebrafish|tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:1|geo loc name:missing|collection date:missing,MB125 2E L6.D709,For Experiment 1 basecall files were converted to FASTQ by bcl2fastq2 demultiplexed quality trimmed and filter using ea utils https://expressionanalysis.github.io/ea utils/ ; last accessed 01 21 2020. Reads with Phred quality scores <7 or >4 Ns at either end of the read were trimmed. Reads with any one base comprising > 95% frequency of the read or having an average Phred quality score <25 were removed. For Experiment 2 Illumina basecall files were converted to FASTQ and demultiplexed by bcl2fastq2. Reads with 3’ Phred quality scores < 20 or lengths <25 nucleotides were trimmed or removed respectively in Partek Flow v.8.0.19.0428; St. Louis MO. Experiments 1 and 2. Trimmed FASTQ files from both sequencing experiments were aligned to Danio rerio GRCz11 using STAR v.2.5.3a with default settings in Partek Flow and strand specificity detection enabled for the paired end reads from Experiment 1 but not for Experiment 2. Gene counts from both experiments were quantified from aligned reads using Partek Expectation Maximization and Ensembl Danio rerio GRCz11.93 gene annotation. Assembly: GRCz11/93 gene annotation Supplementary files format and content: tab delimited file for experiment1 containing raw counts for all samples Supplementary files format and content: tab delimited file for experiment2 containing raw counts for all samples,pooled heads collected anterior to swim bladder of larval zebrafish,,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product’s quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:1,GSM8919412,GSM8919412: MB125 2E L6.D709; Danio rerio; RNA Seq,GSM8919412 r1,GSM8919412,1,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product's quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP579141,,,MB125_2E_L6.D709_1.fastq.gz MB125_2E_L6.D709_2.fastq.gz,fastq fastq,2393568822.0,23466361.0,GSM8919412 r1,0:51 1:51,A:573394367;C:604936257;G:602232683;T:612984188;N:21327,51,51,,,573394367,604936257,602232683,612984188,21327,SRX28428321,SRS24751194,SRA2113523,US EPA,US EPA,,,,,,,,,,,,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2025-04-16,Multi-stage,Multi-stage,Multi-tissue,Multi-system 35905,SRR33167532,SRX28428320,SRS24751192,SRP579141,PRJNA1251588,Investigation into molecular mechanisms behind microbiome and normal neurobehavioral development in zebrafish,GSE294778,Transcriptome Analysis,Changes in resident microbiota may have wide ranging effects on human health. Previous research found axenic zebrafish Danio rerio exhibit hyperactivity at 10 dpf dpf. This abnormal neurobehavioral response can be prevented with re colonization of axenic embryos with microbes at 1 dpf. The present study investigated the molecular mechanism behind this effect through RNA sequencing of pooled 10 dpf larval head tissue from axenic conventionally colonized controls and axenic zebrafish recolonized with microbes at 1 dpf. Overall design: 10 dpf dpf axenic microbe free conventionally colonized normal and axenic Danio rerio embryos recolonized with microbes at 1 dpf control for axenic derivation process were submitted for RNA sequencing to investigate the molecular mechanism behind hyperactivity in axenic larvae.,,,,MB125 2D L6.D710,GSM8919411,,source name:pooled heads collected anterior to swim bladder of larval zebrafish|tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:1|geo loc name:missing|collection date:missing,MB125 2D L6.D710,For Experiment 1 basecall files were converted to FASTQ by bcl2fastq2 demultiplexed quality trimmed and filter using ea utils https://expressionanalysis.github.io/ea utils/ ; last accessed 01 21 2020. Reads with Phred quality scores <7 or >4 Ns at either end of the read were trimmed. Reads with any one base comprising > 95% frequency of the read or having an average Phred quality score <25 were removed. For Experiment 2 Illumina basecall files were converted to FASTQ and demultiplexed by bcl2fastq2. Reads with 3’ Phred quality scores < 20 or lengths <25 nucleotides were trimmed or removed respectively in Partek Flow v.8.0.19.0428; St. Louis MO. Experiments 1 and 2. Trimmed FASTQ files from both sequencing experiments were aligned to Danio rerio GRCz11 using STAR v.2.5.3a with default settings in Partek Flow and strand specificity detection enabled for the paired end reads from Experiment 1 but not for Experiment 2. Gene counts from both experiments were quantified from aligned reads using Partek Expectation Maximization and Ensembl Danio rerio GRCz11.93 gene annotation. Assembly: GRCz11/93 gene annotation Supplementary files format and content: tab delimited file for experiment1 containing raw counts for all samples Supplementary files format and content: tab delimited file for experiment2 containing raw counts for all samples,pooled heads collected anterior to swim bladder of larval zebrafish,,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product’s quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:1,GSM8919411,GSM8919411: MB125 2D L6.D710; Danio rerio; RNA Seq,GSM8919411 r1,GSM8919411,1,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product's quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP579141,,,MB125_2D_L6.D710_1.fastq.gz MB125_2D_L6.D710_2.fastq.gz,fastq fastq,2322596610.0,22770555.0,GSM8919411 r1,0:51 1:51,A:563147167;C:580081508;G:578201930;T:601145790;N:20215,51,51,,,563147167,580081508,578201930,601145790,20215,SRX28428320,SRS24751192,SRA2113523,US EPA,US EPA,,,,,,,,,,,,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2025-04-16,Multi-stage,Multi-stage,Multi-tissue,Multi-system 35906,SRR33167533,SRX28428319,SRS24751193,SRP579141,PRJNA1251588,Investigation into molecular mechanisms behind microbiome and normal neurobehavioral development in zebrafish,GSE294778,Transcriptome Analysis,Changes in resident microbiota may have wide ranging effects on human health. Previous research found axenic zebrafish Danio rerio exhibit hyperactivity at 10 dpf dpf. This abnormal neurobehavioral response can be prevented with re colonization of axenic embryos with microbes at 1 dpf. The present study investigated the molecular mechanism behind this effect through RNA sequencing of pooled 10 dpf larval head tissue from axenic conventionally colonized controls and axenic zebrafish recolonized with microbes at 1 dpf. Overall design: 10 dpf dpf axenic microbe free conventionally colonized normal and axenic Danio rerio embryos recolonized with microbes at 1 dpf control for axenic derivation process were submitted for RNA sequencing to investigate the molecular mechanism behind hyperactivity in axenic larvae.,,,,GRC355 AC1 S4,GSM8919386,,source name:pooled heads collected anterior to swim bladder of larval zebrafish|tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:2|geo loc name:missing|collection date:missing,GRC355 AC1 S4,For Experiment 1 basecall files were converted to FASTQ by bcl2fastq2 demultiplexed quality trimmed and filter using ea utils https://expressionanalysis.github.io/ea utils/ ; last accessed 01 21 2020. Reads with Phred quality scores <7 or >4 Ns at either end of the read were trimmed. Reads with any one base comprising > 95% frequency of the read or having an average Phred quality score <25 were removed. For Experiment 2 Illumina basecall files were converted to FASTQ and demultiplexed by bcl2fastq2. Reads with 3’ Phred quality scores < 20 or lengths <25 nucleotides were trimmed or removed respectively in Partek Flow v.8.0.19.0428; St. Louis MO. Experiments 1 and 2. Trimmed FASTQ files from both sequencing experiments were aligned to Danio rerio GRCz11 using STAR v.2.5.3a with default settings in Partek Flow and strand specificity detection enabled for the paired end reads from Experiment 1 but not for Experiment 2. Gene counts from both experiments were quantified from aligned reads using Partek Expectation Maximization and Ensembl Danio rerio GRCz11.93 gene annotation. Assembly: GRCz11/93 gene annotation Supplementary files format and content: tab delimited file for experiment1 containing raw counts for all samples Supplementary files format and content: tab delimited file for experiment2 containing raw counts for all samples,pooled heads collected anterior to swim bladder of larval zebrafish,,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product’s quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,tissue:pooled heads collected anterior to swim bladder of larval zebrafish|strain:mixed wild type zebrafish line US EPA|lifestage:larvae|age:10 dpf re colonized with microbes on day 1|experiment:2,GSM8919386,GSM8919386: GRC355 AC1 S4; Danio rerio; RNA Seq,GSM8919386 r1,GSM8919386,1,Samples from experiment 1 underwent RNA isolation by Q2 Solutions Expression Analysis LLC Morrisville NC USA. Samples from experiment 2 underwent RNA extraction at US EPA Durham NC USA using Direct zol™ RNA MiniPrep Plus kit Zymo Research per manufacturer instructions. RNA sample quality and concentration from both experiments were checked by Bioanalyzer and Nanodrop or Qubit. For Experiment 1 paired end 50 base pair total RNA sequencing libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo Zero Gold Library Prep Kit for rRNA depletion Illumina San Diego California cat. No. RS 1222303 at Q2 solutions Expression Anaysis according to manufacturer directions. Briefly rRNA was selectively filtered from total RNA samples through streptavidin bead binding and cleanup. Purified RNA then underwent heat fragmentation with divalent cations prior to stranded cDNA synthesis and barcoding of samples using the TruSeq stranded Total RNA kit. cDNA libraries were quantified and cDNA insert size assessed by Agilent Bioanalyzer DNA 1000 kit #5067 1504 and qPCR KAPA Library Quant Kit #KK4824 KAPA Biosystems Wilmington MA. Sample libraries were then normalized randomly pooled and sequenced at 8 samples per lane to a target read depth of >20 million reads on an Illumina HiSeq 2500. For Experiment 2 purified RNA from each sample 500 ng per sample were randomized and processed on Apollo324 for polyA selection and then continued on library prep with PrepX mRNA 48 Protocol v19 at US EPA Durham NC USA. PCR amplification with 32 index primers were run for 13 cycles. The resulting PCR products were cleaned up on Apollo324 with PCR Cleanup 48 Protocol. The volume of purified libraries was about 10 μl. The PCR product's quality was checked on Bioanalyzer with High Sensitivity DNA chips and the average size bp was obtained according to the electropherogram. The collected libraries were quantified by Qubit DNA HS kit. The molar concentration of each library was estimated by using average molecular size from Bioanalyzer data and concentration from Qubit measurement. Then an equal amount from each library μl x nM was pooled from 16 randomized libraries to make the sequencing samples total 2 pools of 16 libraries each and the concentration of the pooled libraries was checked by Qubit. The library pools were denatured and diluted according to Illumina NextSeq protocols. The final concentration for sequencing was 2.5 pM + 2% Phix and it was run for 75 cycles SR Single Read.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP579141,,,GRC355_AC1_S4_R1_001.fastq.gz,fastq,2063624400.0,27514992.0,GSM8919386 r1,0:75,A:560729563;C:466634352;G:528754548;T:507475626;N:30311,75,,,,560729563,466634352,528754548,507475626,30311,SRX28428319,SRS24751193,SRA2113523,US EPA,US EPA,,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,poly_a,trueseq,bulk,bulk,bulk,,United States,2025-04-16,Multi-stage,Multi-stage,Multi-tissue,Multi-system 53513,SRR9906847,SRX6657804,SRS5218265,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdahN36R2,GSM4007833,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:36hr|replicate:Replicate 2,zfRNAnmdahN36R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:36hr|replicate:Replicate 2,GSM4007833,GSM4007833: zfRNAnmdahN36R2; Danio rerio; RNA Seq,GSM4007833,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-TF4N_S8_R1_001.fastq.gz THoa-TF4N_S8_R2_001.fastq.gz,fastq fastq,7928700600.0,52858004.0,GSM4007833 r1,0:75 1:75,A:2214653986;C:1699850211;G:1809132429;T:2204054149;N:1009825,75,75,,,2214653986,1699850211,1809132429,2204054149,1009825,SRX6657804,SRS5218265,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.88014,0.90383,0.394,0.41783,0.76607,0.77043,0.5474,0.55256,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53514,SRR9906846,SRX6657803,SRS5218264,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdahN36R1,GSM4007832,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:36hr|replicate:Replicate 1,zfRNAnmdahN36R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:36hr|replicate:Replicate 1,GSM4007832,GSM4007832: zfRNAnmdahN36R1; Danio rerio; RNA Seq,GSM4007832,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-TF3N_S7_R1_001.fastq.gz THoa-TF3N_S7_R2_001.fastq.gz,fastq fastq,7863596250.0,52423975.0,GSM4007832 r1,0:75 1:75,A:2245214380;C:1641445245;G:1740385747;T:2235554179;N:996699,75,75,,,2245214380,1641445245,1740385747,2235554179,996699,SRX6657803,SRS5218264,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.86899,0.89643,0.43639,0.46473,0.76631,0.77086,0.53259,0.53462,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53515,SRR9906845,SRX6657802,SRS5218263,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdahN20R2,GSM4007831,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:20hr|replicate:Replicate 2,zfRNAnmdahN20R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:20hr|replicate:Replicate 2,GSM4007831,GSM4007831: zfRNAnmdahN20R2; Danio rerio; RNA Seq,GSM4007831,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-TF2N_S6_R1_001.fastq.gz THoa-TF2N_S6_R2_001.fastq.gz,fastq fastq,11221029450.0,74806863.0,GSM4007831 r1,0:75 1:75,A:3206652122;C:2349148827;G:2475237997;T:3188565893;N:1424611,75,75,,,3206652122,2349148827,2475237997,3188565893,1424611,SRX6657802,SRS5218263,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.86571,0.89458,0.44089,0.4703,0.77163,0.77573,0.54887,0.54576,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53516,SRR9906844,SRX6657801,SRS5218262,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdahN20R1,GSM4007830,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:20hr|replicate:Replicate 1,zfRNAnmdahN20R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:20hr|replicate:Replicate 1,GSM4007830,GSM4007830: zfRNAnmdahN20R1; Danio rerio; RNA Seq,GSM4007830,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-TF1N_S5_R1_001.fastq.gz THoa-TF1N_S5_R2_001.fastq.gz,fastq fastq,11263633950.0,75090893.0,GSM4007830 r1,0:75 1:75,A:3198241763;C:2374935676;G:2514695492;T:3174314457;N:1446562,75,75,,,3198241763,2374935676,2514695492,3174314457,1446562,SRX6657801,SRS5218262,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.87624,0.90304,0.39909,0.42417,0.76984,0.77376,0.53406,0.53718,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53517,SRR9906843,SRX6657800,SRS5218261,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdahP36R2,GSM4007829,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:36hr|replicate:Replicate 2,zfRNAnmdahP36R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:36hr|replicate:Replicate 2,GSM4007829,GSM4007829: zfRNAnmdahP36R2; Danio rerio; RNA Seq,GSM4007829,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-TF4P_S4_R1_001.fastq.gz THoa-TF4P_S4_R2_001.fastq.gz,fastq fastq,10599378900.0,70662526.0,GSM4007829 r1,0:75 1:75,A:2917939167;C:2338922409;G:2472546084;T:2868616483;N:1354757,75,75,,,2917939167,2338922409,2472546084,2868616483,1354757,SRX6657800,SRS5218261,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.89693,0.9159,0.31259,0.33022,0.7371,0.74182,0.56412,0.56559,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53518,SRR9906842,SRX6657799,SRS5218260,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdahP36R1,GSM4007828,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:36hr|replicate:Replicate 1,zfRNAnmdahP36R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:36hr|replicate:Replicate 1,GSM4007828,GSM4007828: zfRNAnmdahP36R1; Danio rerio; RNA Seq,GSM4007828,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-TF3P_S3_R1_001.fastq.gz THoa-TF3P_S3_R2_001.fastq.gz,fastq fastq,11034687150.0,73564581.0,GSM4007828 r1,0:75 1:75,A:2954740084;C:2509220120;G:2662792371;T:2906521814;N:1412761,75,75,,,2954740084,2509220120,2662792371,2906521814,1412761,SRX6657799,SRS5218260,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.90866,0.9253,0.29649,0.3138,0.7419,0.74793,0.57845,0.59013,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53519,SRR9906841,SRX6657798,SRS5218259,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdahP20R2,GSM4007827,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:20hr|replicate:Replicate 2,zfRNAnmdahP20R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:20hr|replicate:Replicate 2,GSM4007827,GSM4007827: zfRNAnmdahP20R2; Danio rerio; RNA Seq,GSM4007827,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-TF2P_S2_R1_001.fastq.gz THoa-TF2P_S2_R2_001.fastq.gz,fastq fastq,10416796950.0,69445313.0,GSM4007827 r1,0:75 1:75,A:2846027907;C:2324721617;G:2438862539;T:2805866203;N:1318684,75,75,,,2846027907,2324721617,2438862539,2805866203,1318684,SRX6657798,SRS5218259,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.89999,0.92352,0.30772,0.32822,0.74823,0.75089,0.5489,0.55828,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53520,SRR9906840,SRX6657797,SRS5218258,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdahP20R1,GSM4007826,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:20hr|replicate:Replicate 1,zfRNAnmdahP20R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:20hr|replicate:Replicate 1,GSM4007826,GSM4007826: zfRNAnmdahP20R1; Danio rerio; RNA Seq,GSM4007826,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-TF1P_S1_R1_001.fastq.gz THoa-TF1P_S1_R2_001.fastq.gz,fastq fastq,11087990850.0,73919939.0,GSM4007826 r1,0:75 1:75,A:3086331907;C:2414289581;G:2547013161;T:3038947630;N:1408571,75,75,,,3086331907,2414289581,2547013161,3038947630,1408571,SRX6657797,SRS5218258,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.89541,0.9175,0.30129,0.32289,0.7417,0.74714,0.55292,0.55122,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53521,SRR9906839,SRX6657796,SRS5218257,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdaN36R2,GSM4007825,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:36hr|replicate:Replicate 2,zfRNAnmdaN36R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:36hr|replicate:Replicate 2,GSM4007825,GSM4007825: zfRNAnmdaN36R2; Danio rerio; RNA Seq,GSM4007825,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF16N_S7_R1_001.fastq.gz THoa-DF16N_S7_R2_001.fastq.gz,fastq fastq,7593295650.0,50621971.0,GSM4007825 r1,0:75 1:75,A:1842742463;C:1931868500;G:2009769570;T:1805690959;N:3224158,75,75,,,1842742463,1931868500,2009769570,1805690959,3224158,SRX6657796,SRS5218257,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.93981,0.95204,0.24731,0.26265,0.81412,0.81811,0.67154,0.67196,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53522,SRR9906838,SRX6657795,SRS5218256,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdaN36R1,GSM4007824,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:36hr|replicate:Replicate 1,zfRNAnmdaN36R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:36hr|replicate:Replicate 1,GSM4007824,GSM4007824: zfRNAnmdaN36R1; Danio rerio; RNA Seq,GSM4007824,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF15N_S6_R1_001.fastq.gz THoa-DF15N_S6_R2_001.fastq.gz,fastq fastq,7927875450.0,52852503.0,GSM4007824 r1,0:75 1:75,A:1938512738;C:1988054202;G:2080463145;T:1917497438;N:3347927,75,75,,,1938512738,1988054202,2080463145,1917497438,3347927,SRX6657795,SRS5218256,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.93468,0.94808,0.25462,0.27334,0.81262,0.81548,0.6351,0.66108,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53523,SRR9906837,SRX6657794,SRS5218255,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdaN20R2,GSM4007823,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:20hr|replicate:Replicate 2,zfRNAnmdaN20R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:20hr|replicate:Replicate 2,GSM4007823,GSM4007823: zfRNAnmdaN20R2; Danio rerio; RNA Seq,GSM4007823,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,DF29N_S7_R1_001.fastq.gz DF29N_S7_R2_001.fastq.gz,fastq fastq,7876840694.0,52829905.0,GSM4007823 r1,0:74.55 1:74.55,A:2010274884;C:1895760244;G:1963812981;T:2004863879;N:2128706,74,74,,,2010274884,1895760244,1963812981,2004863879,2128706,SRX6657794,SRS5218255,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.90858,0.91979,0.28522,0.29978,0.81742,0.82104,0.6582,0.65941,74,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53524,SRR9906836,SRX6657793,SRS5218254,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdaN20R1,GSM4007822,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:20hr|replicate:Replicate 1,zfRNAnmdaN20R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:20hr|replicate:Replicate 1,GSM4007822,GSM4007822: zfRNAnmdaN20R1; Danio rerio; RNA Seq,GSM4007822,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF13N_S5_R1_001.fastq.gz THoa-DF13N_S5_R2_001.fastq.gz,fastq fastq,8003905950.0,53359373.0,GSM4007822 r1,0:75 1:75,A:1925414212;C:2030985118;G:2137411382;T:1906727349;N:3367889,75,75,,,1925414212,2030985118,2137411382,1906727349,3367889,SRX6657793,SRS5218254,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.94476,0.95272,0.22584,0.24304,0.81404,0.8186,0.68349,0.6811,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53525,SRR9906835,SRX6657792,SRS5218253,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdaN10R2,GSM4007821,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:10hr|replicate:Replicate 2,zfRNAnmdaN10R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:10hr|replicate:Replicate 2,GSM4007821,GSM4007821: zfRNAnmdaN10R2; Danio rerio; RNA Seq,GSM4007821,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF12N_S4_R1_001.fastq.gz THoa-DF12N_S4_R2_001.fastq.gz,fastq fastq,7571445300.0,50476302.0,GSM4007821 r1,0:75 1:75,A:1961355978;C:1793618129;G:1874282399;T:1938980874;N:3207920,75,75,,,1961355978,1793618129,1874282399,1938980874,3207920,SRX6657792,SRS5218253,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.91789,0.93655,0.28528,0.30489,0.7989,0.80079,0.6136,0.62523,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53526,SRR9906834,SRX6657791,SRS5218252,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdaN10R1,GSM4007820,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:10hr|replicate:Replicate 1,zfRNAnmdaN10R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:10hr|replicate:Replicate 1,GSM4007820,GSM4007820: zfRNAnmdaN10R1; Danio rerio; RNA Seq,GSM4007820,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF11N_S3_R1_001.fastq.gz THoa-DF11N_S3_R2_001.fastq.gz,fastq fastq,6515556150.0,43437041.0,GSM4007820 r1,0:75 1:75,A:1542198255;C:1688374350;G:1771407578;T:1510810367;N:2765600,75,75,,,1542198255,1688374350,1771407578,1510810367,2765600,SRX6657791,SRS5218252,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.94732,0.95644,0.21363,0.23778,0.82698,0.83019,0.69676,0.69466,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53527,SRR9906833,SRX6657790,SRS5218251,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdaN04R2,GSM4007819,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:4hr|replicate:Replicate 2,zfRNAnmdaN04R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:4hr|replicate:Replicate 2,GSM4007819,GSM4007819: zfRNAnmdaN04R2; Danio rerio; RNA Seq,GSM4007819,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF10N_S2_R1_001.fastq.gz THoa-DF10N_S2_R2_001.fastq.gz,fastq fastq,7522510200.0,50150068.0,GSM4007819 r1,0:75 1:75,A:1828130590;C:1927363004;G:1976726604;T:1787097599;N:3192403,75,75,,,1828130590,1927363004,1976726604,1787097599,3192403,SRX6657790,SRS5218251,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.94502,0.9468,0.20905,0.22188,0.8436,0.8454,0.72316,0.72635,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53528,SRR9906832,SRX6657789,SRS5218250,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdaN04R1,GSM4007818,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:4hr|replicate:Replicate 1,zfRNAnmdaN04R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP |time:4hr|replicate:Replicate 1,GSM4007818,GSM4007818: zfRNAnmdaN04R1; Danio rerio; RNA Seq,GSM4007818,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF9N_S1_R1_001.fastq.gz THoa-DF9N_S1_R2_001.fastq.gz,fastq fastq,7392867750.0,49285785.0,GSM4007818 r1,0:75 1:75,A:1807364965;C:1871069705;G:1942445368;T:1768857698;N:3130014,75,75,,,1807364965,1871069705,1942445368,1768857698,3130014,SRX6657789,SRS5218250,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.93966,0.94358,0.23683,0.25285,0.81785,0.82012,0.67242,0.69507,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53529,SRR9906831,SRX6657788,SRS5218249,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdaP36R2,GSM4007817,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:36hr|replicate:Replicate 2,zfRNAnmdaP36R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:36hr|replicate:Replicate 2,GSM4007817,GSM4007817: zfRNAnmdaP36R2; Danio rerio; RNA Seq,GSM4007817,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF16P_S7_R1_001.fastq.gz THoa-DF16P_S7_R2_001.fastq.gz,fastq fastq,6373735248.0,42722100.0,GSM4007817 r1,0:74.59 1:74.60,A:1515782870;C:1648277663;G:1705206622;T:1501843556;N:2624537,74,74,,,1515782870,1648277663,1705206622,1501843556,2624537,SRX6657788,SRS5218249,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.94124,0.94458,0.14833,0.15451,0.82012,0.8239,0.6622,0.67093,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53530,SRR9906830,SRX6657787,SRS5218248,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdaP36R1,GSM4007816,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:36hr|replicate:Replicate 1,zfRNAnmdaP36R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:36hr|replicate:Replicate 1,GSM4007816,GSM4007816: zfRNAnmdaP36R1; Danio rerio; RNA Seq,GSM4007816,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF15P_S6_R1_001.fastq.gz THoa-DF15P_S6_R2_001.fastq.gz,fastq fastq,6569582369.0,44039507.0,GSM4007816 r1,0:74.58 1:74.59,A:1581167169;C:1668076484;G:1734283999;T:1583362809;N:2691908,74,74,,,1581167169,1668076484,1734283999,1583362809,2691908,SRX6657787,SRS5218248,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.92986,0.93029,0.15525,0.16583,0.80825,0.81172,0.65632,0.67275,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53531,SRR9906829,SRX6657786,SRS5218247,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdaP20R2R2,GSM4007815,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:20hr|replicate:Replicate 2,zfRNAnmdaP20R2R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:20hr|replicate:Replicate 2,GSM4007815,GSM4007815: zfRNAnmdaP20R2R2; Danio rerio; RNA Seq,GSM4007815,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF29P-run2_S7_R1_001.fastq.gz THoa-DF29P-run2_S7_R2_001.fastq.gz,fastq fastq,4138950900.0,27593006.0,GSM4007815 r1,0:75 1:75,A:1036118589;C:1022903513;G:1048995320;T:1027851668;N:3081810,75,75,,,1036118589,1022903513,1048995320,1027851668,3081810,SRX6657786,SRS5218247,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.94004,0.94676,0.14485,0.14607,0.80986,0.8102,0.6594,0.60583,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53532,SRR9906828,SRX6657785,SRS5218246,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdaP20R2R1,GSM4007814,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:20hr|replicate:Replicate 2,zfRNAnmdaP20R2R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:20hr|replicate:Replicate 2,GSM4007814,GSM4007814: zfRNAnmdaP20R2R1; Danio rerio; RNA Seq,GSM4007814,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF29P-run1_S7_R1_001.fastq.gz THoa-DF29P-run1_S7_R2_001.fastq.gz,fastq fastq,2345611650.0,15637411.0,GSM4007814 r1,0:75 1:75,A:586100477;C:579733712;G:596500443;T:582730098;N:546920,75,75,,,586100477,579733712,596500443,582730098,546920,SRX6657785,SRS5218246,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.93951,0.94484,0.14437,0.14581,0.80738,0.80724,0.66526,0.58765,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53533,SRR9906827,SRX6657784,SRS5218245,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdaP20R1,GSM4007813,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:20hr|replicate:Replicate 1,zfRNAnmdaP20R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:20hr|replicate:Replicate 1,GSM4007813,GSM4007813: zfRNAnmdaP20R1; Danio rerio; RNA Seq,GSM4007813,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF13P_S5_R1_001.fastq.gz THoa-DF13P_S5_R2_001.fastq.gz,fastq fastq,7039003072.0,47187560.0,GSM4007813 r1,0:74.58 1:74.59,A:1712945752;C:1757482359;G:1842953199;T:1722863594;N:2758168,74,74,,,1712945752,1757482359,1842953199,1722863594,2758168,SRX6657784,SRS5218245,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.94233,0.94816,0.14168,0.15153,0.79977,0.80549,0.62578,0.64305,72,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53534,SRR9906826,SRX6657783,SRS5218244,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdaP10R2,GSM4007812,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:10hr|replicate:Replicate 2,zfRNAnmdaP10R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:10hr|replicate:Replicate 2,GSM4007812,GSM4007812: zfRNAnmdaP10R2; Danio rerio; RNA Seq,GSM4007812,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF12P_S4_R1_001.fastq.gz THoa-DF12P_S4_R2_001.fastq.gz,fastq fastq,5745697684.0,38525032.0,GSM4007812 r1,0:74.57 1:74.57,A:1416523959;C:1432155681;G:1476691642;T:1417880795;N:2445607,74,74,,,1416523959,1432155681,1476691642,1417880795,2445607,SRX6657783,SRS5218244,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.93423,0.94349,0.1837,0.191,0.81146,0.81446,0.64969,0.65118,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53535,SRR9906825,SRX6657782,SRS5218243,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdaP10R1,GSM4007811,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:10hr|replicate:Replicate 1,zfRNAnmdaP10R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:10hr|replicate:Replicate 1,GSM4007811,GSM4007811: zfRNAnmdaP10R1; Danio rerio; RNA Seq,GSM4007811,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF11P_S3_R1_001.fastq.gz THoa-DF11P_S3_R2_001.fastq.gz,fastq fastq,4687597951.0,31430998.0,GSM4007811 r1,0:74.56 1:74.58,A:1186920007;C:1133558971;G:1178442772;T:1186839573;N:1836628,74,74,,,1186920007,1133558971,1178442772,1186839573,1836628,SRX6657782,SRS5218243,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.93636,0.94612,0.16988,0.17882,0.80014,0.80424,0.63955,0.63931,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53536,SRR9906824,SRX6657781,SRS5218242,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdaP04R2,GSM4007810,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:4hr|replicate:Replicate 2,zfRNAnmdaP04R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:4hr|replicate:Replicate 2,GSM4007810,GSM4007810: zfRNAnmdaP04R2; Danio rerio; RNA Seq,GSM4007810,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF10P_S2_R1_001.fastq.gz THoa-DF10P_S2_R2_001.fastq.gz,fastq fastq,5482033611.0,36761528.0,GSM4007810 r1,0:74.56 1:74.57,A:1399163955;C:1327953597;G:1360796447;T:1391846223;N:2273389,74,74,,,1399163955,1327953597,1360796447,1391846223,2273389,SRX6657781,SRS5218242,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.86015,0.87027,0.18565,0.19683,0.83349,0.83666,0.62827,0.63846,72,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53537,SRR9906823,SRX6657780,SRS5218241,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAnmdaP04R1,GSM4007809,,source name:Retina|tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:4hr|replicate:Replicate 1,zfRNAnmdaP04R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:GFP]mi2001 AB|condition:NMDA treatment|cell population of facs sorting:GFP+|time:4hr|replicate:Replicate 1,GSM4007809,GSM4007809: zfRNAnmdaP04R1; Danio rerio; RNA Seq,GSM4007809,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF9P_S1_R1_001.fastq.gz THoa-DF9P_S1_R2_001.fastq.gz,fastq fastq,21735411065.0,145822702.0,GSM4007809 r1,0:74.54 1:74.51,A:5770843248;C:4924186554;G:5223386393;T:5808415210;N:8579660,74,74,,,5770843248,4924186554,5223386393,5808415210,8579660,SRX6657780,SRS5218241,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.32804,0.332,0.08428,0.08869,0.87584,0.87969,0.63905,0.65793,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53538,SRR9906822,SRX6657779,SRS5218240,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldtN36R2,GSM4007808,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP |time:36hr|replicate:Replicate 2,zfRNAldtN36R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP |time:36hr|replicate:Replicate 2,GSM4007808,GSM4007808: zfRNAldtN36R2; Danio rerio; RNA Seq,GSM4007808,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF8N_S9_R1_001.fastq.gz THoa-DF8N_S9_R2_001.fastq.gz,fastq fastq,7461557400.0,49743716.0,GSM4007808 r1,0:75 1:75,A:1829397197;C:1872660811;G:1943038567;T:1813306644;N:3154181,75,75,,,1829397197,1872660811,1943038567,1813306644,3154181,SRX6657779,SRS5218240,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.9181,0.93155,0.26114,0.27679,0.80152,0.8045,0.67335,0.67474,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53539,SRR9906821,SRX6657778,SRS5218239,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldtN36R1,GSM4007807,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP |time:36hr|replicate:Replicate 1,zfRNAldtN36R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP |time:36hr|replicate:Replicate 1,GSM4007807,GSM4007807: zfRNAldtN36R1; Danio rerio; RNA Seq,GSM4007807,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF7N_S8_R1_001.fastq.gz THoa-DF7N_S8_R2_001.fastq.gz,fastq fastq,7310480700.0,48736538.0,GSM4007807 r1,0:75 1:75,A:1851060267;C:1761439990;G:1858394380;T:1836486630;N:3099433,75,75,,,1851060267,1761439990,1858394380,1836486630,3099433,SRX6657778,SRS5218239,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.92044,0.93499,0.28396,0.29825,0.77948,0.7825,0.62758,0.63403,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53540,SRR9906820,SRX6657777,SRS5218238,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldtN20R2,GSM4007806,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP |time:20hr|replicate:Replicate 2,zfRNAldtN20R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP |time:20hr|replicate:Replicate 2,GSM4007806,GSM4007806: zfRNAldtN20R2; Danio rerio; RNA Seq,GSM4007806,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF6N_S9_R1_001.fastq.gz THoa-DF6N_S9_R2_001.fastq.gz,fastq fastq,7098730131.0,47579801.0,GSM4007806 r1,0:74.60 1:74.59,A:1717050162;C:1797575844;G:1860363186;T:1720884708;N:2856231,74,74,,,1717050162,1797575844,1860363186,1720884708,2856231,SRX6657777,SRS5218238,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.93627,0.93912,0.23916,0.25175,0.80945,0.81418,0.67342,0.68259,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53541,SRR9906819,SRX6657776,SRS5218237,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldtN20R1,GSM4007805,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP |time:20hr|replicate:Replicate 1,zfRNAldtN20R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP |time:20hr|replicate:Replicate 1,GSM4007805,GSM4007805: zfRNAldtN20R1; Danio rerio; RNA Seq,GSM4007805,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF5N_S8_R1_001.fastq.gz THoa-DF5N_S8_R2_001.fastq.gz,fastq fastq,7507942092.0,50334342.0,GSM4007805 r1,0:74.59 1:74.57,A:1866081920;C:1827112880;G:1922774342;T:1888960357;N:3012593,74,74,,,1866081920,1827112880,1922774342,1888960357,3012593,SRX6657776,SRS5218237,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.92572,0.93469,0.27326,0.28815,0.79084,0.79707,0.61558,0.63121,75,74,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53542,SRR9906818,SRX6657775,SRS5218236,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldtN10R2,GSM4007804,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP |time:10hr|replicate:Replicate 2,zfRNAldtN10R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP |time:10hr|replicate:Replicate 2,GSM4007804,GSM4007804: zfRNAldtN10R2; Danio rerio; RNA Seq,GSM4007804,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF4N_S4_R1_001.fastq.gz THoa-DF4N_S4_R2_001.fastq.gz,fastq fastq,9118674847.0,60348350.0,GSM4007804 r1,0:75.55 1:75.55,A:2354247285;C:2165731401;G:2244032848;T:2353621167;N:1042146,75,75,,,2354247285,2165731401,2244032848,2353621167,1042146,SRX6657775,SRS5218236,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.90175,0.91595,0.25813,0.27158,0.79125,0.7931,0.62118,0.61894,76,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53543,SRR9906817,SRX6657774,SRS5218235,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldtN10R1R2,GSM4007803,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP |time:10hr|replicate:Replicate 1,zfRNAldtN10R1R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP |time:10hr|replicate:Replicate 1,GSM4007803,GSM4007803: zfRNAldtN10R1R2; Danio rerio; RNA Seq,GSM4007803,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF2N_S4_R1_001.fastq.gz THoa-DF2N_S4_R2_001.fastq.gz,fastq fastq,6920389304.0,45528877.0,GSM4007803 r1,0:76 1:76,A:1832467265;C:1590107325;G:1661000950;T:1836680423;N:133341,76,76,,,1832467265,1590107325,1661000950,1836680423,133341,SRX6657774,SRS5218235,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.89667,0.91615,0.32531,0.34748,0.78407,0.78628,0.61109,0.59996,76,76,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53544,SRR9906816,SRX6657773,SRS5218234,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldtN10R1R1,GSM4007802,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP |time:10hr|replicate:Replicate 1,zfRNAldtN10R1R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP |time:10hr|replicate:Replicate 1,GSM4007802,GSM4007802: zfRNAldtN10R1R1; Danio rerio; RNA Seq,GSM4007802,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,DF2N_S4_R1_001.fastq.gz DF2N_S4_R2_001.fastq.gz,fastq fastq,6701567912.0,44400966.0,GSM4007802 r1,0:75.49 1:75.44,A:1688384411;C:1625844846;G:1698926520;T:1686384951;N:2027184,75,75,,,1688384411,1625844846,1698926520,1686384951,2027184,SRX6657773,SRS5218234,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.91379,0.93036,0.29573,0.31837,0.80034,0.8017,0.638,0.65469,76,76,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53545,SRR9906815,SRX6657772,SRS5218233,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldtN04R2,GSM4007801,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP |time:4hr|replicate:Replicate 2,zfRNAldtN04R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP |time:4hr|replicate:Replicate 2,GSM4007801,GSM4007801: zfRNAldtN04R2; Danio rerio; RNA Seq,GSM4007801,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF3N_S3_R1_001.fastq.gz THoa-DF3N_S3_R2_001.fastq.gz,fastq fastq,9452762624.0,62556061.0,GSM4007801 r1,0:75.55 1:75.56,A:2418410246;C:2278741513;G:2354276985;T:2400236180;N:1097700,75,75,,,2418410246,2278741513,2354276985,2400236180,1097700,SRX6657772,SRS5218233,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.90533,0.91159,0.20572,0.21726,0.81349,0.81436,0.6425,0.64845,75,76,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53546,SRR9906814,SRX6657771,SRS5218232,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldtN04R1R2,GSM4007800,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP |time:4hr|replicate:Replicate 1,zfRNAldtN04R1R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP |time:4hr|replicate:Replicate 1,GSM4007800,GSM4007800: zfRNAldtN04R1R2; Danio rerio; RNA Seq,GSM4007800,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF1N_S3_R1_001.fastq.gz THoa-DF1N_S3_R2_001.fastq.gz,fastq fastq,6443366488.0,42390569.0,GSM4007800 r1,0:76 1:76,A:1668027282;C:1512803937;G:1592154543;T:1670256264;N:124462,76,76,,,1668027282,1512803937,1592154543,1670256264,124462,SRX6657771,SRS5218232,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.90685,0.92348,0.30206,0.32259,0.80016,0.80156,0.63847,0.61929,76,76,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53547,SRR9906813,SRX6657770,SRS5218231,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldtP36R2R2,GSM4007799,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:36hr|replicate:Replicate 2,zfRNAldtP36R2R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:36hr|replicate:Replicate 2,GSM4007799,GSM4007799: zfRNAldtP36R2R2; Danio rerio; RNA Seq,GSM4007799,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF8P_S9_R1_001.fastq.gz THoa-DF8P_S9_R2_001.fastq.gz,fastq fastq,6236494032.0,41029566.0,GSM4007799 r1,0:76 1:76,A:1465414386;C:1634912062;G:1681819477;T:1454230315;N:117792,76,76,,,1465414386,1634912062,1681819477,1454230315,117792,SRX6657770,SRS5218231,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.94408,0.94952,0.15494,0.16176,0.82404,0.82544,0.67236,0.65741,76,76,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53548,SRR9906812,SRX6657769,SRS5218230,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldtP36R1R2,GSM4007798,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:36hr|replicate:Replicate 1,zfRNAldtP36R1R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:36hr|replicate:Replicate 1,GSM4007798,GSM4007798: zfRNAldtP36R1R2; Danio rerio; RNA Seq,GSM4007798,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF7P_S10_R1_001.fastq.gz THoa-DF7P_S10_R2_001.fastq.gz,fastq fastq,7484077973.0,50162298.0,GSM4007798 r1,0:74.59 1:74.61,A:1782486863;C:1922264544;G:1989068688;T:1787280217;N:2977661,74,74,,,1782486863,1922264544,1989068688,1787280217,2977661,SRX6657769,SRS5218230,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.95471,0.95346,0.15186,0.1567,0.80332,0.80736,0.64996,0.66954,74,74,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53549,SRR9906811,SRX6657768,SRS5218229,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldtP20R3,GSM4007797,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:20hr|replicate:Replicate 2,zfRNAldtP20R3,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:20hr|replicate:Replicate 2,GSM4007797,GSM4007797: zfRNAldtP20R3; Danio rerio; RNA Seq,GSM4007797,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,DF31P_S8_R1_001.fastq.gz DF31P_S8_R2_001.fastq.gz,fastq fastq,8153845075.0,54684638.0,GSM4007797 r1,0:74.54 1:74.57,A:2035354223;C:2023329236;G:2076788826;T:2016246616;N:2126174,74,74,,,2035354223,2023329236,2076788826,2016246616,2126174,SRX6657768,SRS5218229,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.93265,0.94088,0.15684,0.16053,0.80846,0.81044,0.67966,0.68311,75,72,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53550,SRR9906810,SRX6657767,SRS5218228,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldtP20R1R2,GSM4007796,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:20hr|replicate:Replicate 1,zfRNAldtP20R1R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:20hr|replicate:Replicate 1,GSM4007796,GSM4007796: zfRNAldtP20R1R2; Danio rerio; RNA Seq,GSM4007796,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF5P_S7_R1_001.fastq.gz THoa-DF5P_S7_R2_001.fastq.gz,fastq fastq,7112121496.0,46790273.0,GSM4007796 r1,0:76 1:76,A:1739973400;C:1792717453;G:1855648989;T:1723646755;N:134899,76,76,,,1739973400,1792717453,1855648989,1723646755,134899,SRX6657767,SRS5218228,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.879,0.88409,0.13484,0.14289,0.83542,0.83646,0.67507,0.70811,76,76,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53551,SRR9906809,SRX6657766,SRS5218227,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldtP10R3R2,GSM4007795,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:10hr|replicate:Replicate 2,zfRNAldtP10R3R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:10hr|replicate:Replicate 2,GSM4007795,GSM4007795: zfRNAldtP10R3R2; Danio rerio; RNA Seq,GSM4007795,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,Thoa-DF30P-run2_S8_R1_001.fastq.gz Thoa-DF30P-run2_S8_R2_001.fastq.gz,fastq fastq,12756789300.0,85045262.0,GSM4007795 r1,0:75 1:75,A:3411640312;C:2880127219;G:3046896925;T:3408585301;N:9539543,75,75,,,3411640312,2880127219,3046896925,3408585301,9539543,SRX6657766,SRS5218227,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.33812,0.34433,0.0768,0.0802,0.86082,0.86058,0.65811,0.65755,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53552,SRR9906808,SRX6657765,SRS5218226,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldtP10R3R1,GSM4007794,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:10hr|replicate:Replicate 2,zfRNAldtP10R3R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:10hr|replicate:Replicate 2,GSM4007794,GSM4007794: zfRNAldtP10R3R1; Danio rerio; RNA Seq,GSM4007794,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF30P-run1_S8_R1_001.fastq.gz THoa-DF30P-run1_S8_R2_001.fastq.gz,fastq fastq,7263559800.0,48423732.0,GSM4007794 r1,0:75 1:75,A:1936778022;C:1641879024;G:1743981633;T:1939224391;N:1696730,75,75,,,1936778022,1641879024,1743981633,1939224391,1696730,SRX6657765,SRS5218226,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.33555,0.34158,0.07618,0.07959,0.85847,0.8594,0.66386,0.65576,75,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53553,SRR9906807,SRX6657764,SRS5218225,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldtP10R1R2,GSM4007793,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:10hr|replicate:Replicate 1,zfRNAldtP10R1R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:10hr|replicate:Replicate 1,GSM4007793,GSM4007793: zfRNAldtP10R1R2; Danio rerio; RNA Seq,GSM4007793,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF2P_S2_R1_001.fastq.gz THoa-DF2P_S2_R2_001.fastq.gz,fastq fastq,6707936472.0,44131161.0,GSM4007793 r1,0:76 1:76,A:1740799990;C:1575292568;G:1651597418;T:1740118005;N:128491,76,76,,,1740799990,1575292568,1651597418,1740118005,128491,SRX6657764,SRS5218225,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.8382,0.85278,0.22937,0.24498,0.79046,0.79228,0.65323,0.64817,76,76,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53554,SRR9906806,SRX6657763,SRS5218224,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldtP04R2R2,GSM4007792,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:4hr|replicate:Replicate 2,zfRNAldtP04R2R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:4hr|replicate:Replicate 2,GSM4007792,GSM4007792: zfRNAldtP04R2R2; Danio rerio; RNA Seq,GSM4007792,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF3P_S5_R1_001.fastq.gz THoa-DF3P_S5_R2_001.fastq.gz,fastq fastq,6486340688.0,42673294.0,GSM4007792 r1,0:76 1:76,A:1620859265;C:1595766403;G:1651838644;T:1617753810;N:122566,76,76,,,1620859265,1595766403,1651838644,1617753810,122566,SRX6657763,SRS5218224,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.9266,0.93745,0.22272,0.23594,0.80693,0.80886,0.66624,0.66443,76,76,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53555,SRR9906805,SRX6657762,SRS5218223,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldtP04R2R1,GSM4007791,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:4hr|replicate:Replicate 2,zfRNAldtP04R2R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:4hr|replicate:Replicate 2,GSM4007791,GSM4007791: zfRNAldtP04R2R1; Danio rerio; RNA Seq,GSM4007791,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,DF3P_S5_R1_001.fastq.gz DF3P_S5_R2_001.fastq.gz,fastq fastq,6232060716.0,41263515.0,GSM4007791 r1,0:75.53 1:75.50,A:1454262167;C:1640522906;G:1684030380;T:1451528226;N:1717037,75,75,,,1454262167,1640522906,1684030380,1451528226,1717037,SRX6657762,SRS5218223,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.94564,0.95222,0.21542,0.23023,0.8339,0.83707,0.69637,0.6205,76,75,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53556,SRR9906804,SRX6657761,SRS5218222,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldtP04R1R2,GSM4007790,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:4hr|replicate:Replicate 1,zfRNAldtP04R1R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:4hr|replicate:Replicate 1,GSM4007790,GSM4007790: zfRNAldtP04R1R2; Danio rerio; RNA Seq,GSM4007790,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-DF1P_S1_R1_001.fastq.gz THoa-DF1P_S1_R2_001.fastq.gz,fastq fastq,7005274768.0,46087334.0,GSM4007790 r1,0:76 1:76,A:1776496242;C:1692239427;G:1767588879;T:1768815957;N:134263,76,76,,,1776496242,1692239427,1767588879,1768815957,134263,SRX6657761,SRS5218222,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.90794,0.9194,0.26514,0.28336,0.81077,0.81124,0.63299,0.65518,76,76,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53557,SRR9906803,SRX6657760,SRS5218221,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldtP04R1R1,GSM4007789,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:4hr|replicate:Replicate 1,zfRNAldtP04R1R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:4hr|replicate:Replicate 1,GSM4007789,GSM4007789: zfRNAldtP04R1R1; Danio rerio; RNA Seq,GSM4007789,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,DF1P_S1_R1_001.fastq.gz DF1P_S1_R2_001.fastq.gz,fastq fastq,6061320621.0,40165032.0,GSM4007789 r1,0:75.46 1:75.45,A:1512598083;C:1489834050;G:1544112556;T:1512543832;N:2232100,75,75,,,1512598083,1489834050,1544112556,1512543832,2232100,SRX6657760,SRS5218221,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.88455,0.89692,0.25418,0.26915,0.82625,0.82769,0.65697,0.64732,76,76,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53558,SRR9906802,SRX6657759,SRS5218220,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAadtN00R2,GSM4007788,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Control|cell population of facs sorting:GFP |time:Adult|replicate:Replicate 2,zfRNAadtN00R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Control|cell population of facs sorting:GFP |time:Adult|replicate:Replicate 2,GSM4007788,GSM4007788: zfRNAadtN00R2; Danio rerio; RNA Seq,GSM4007788,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-N2_S2_R1_001.fastq.gz THoa-N2_S2_R2_001.fastq.gz,fastq fastq,8845507010.0,58568328.0,GSM4007788 r1,0:75.51 1:75.51,A:2454613681;C:1922905558;G:2022484926;T:2444448951;N:1053894,75,75,,,2454613681,1922905558,2022484926,2444448951,1053894,SRX6657759,SRS5218220,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.89621,0.91455,0.27054,0.28804,0.79703,0.79906,0.59135,0.58068,75,76,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53559,SRR9906801,SRX6657758,SRS5218219,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAadtN00R1,GSM4007787,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Control|cell population of facs sorting:GFP |time:Adult|replicate:Replicate 1,zfRNAadtN00R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Control|cell population of facs sorting:GFP |time:Adult|replicate:Replicate 1,GSM4007787,GSM4007787: zfRNAadtN00R1; Danio rerio; RNA Seq,GSM4007787,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,THoa-N1_S1_R1_001.fastq.gz THoa-N1_S1_R2_001.fastq.gz,fastq fastq,8187702247.0,54214303.0,GSM4007787 r1,0:75.52 1:75.51,A:2264577330;C:1793888388;G:1895064861;T:2233150910;N:1020758,75,75,,,2264577330,1793888388,1895064861,2233150910,1020758,SRX6657758,SRS5218219,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.88635,0.90124,0.22483,0.23897,0.81237,0.81568,0.55133,0.54969,76,76,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53560,SRR9906800,SRX6657757,SRS5218218,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAadtP00R2,GSM4007786,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Control|cell population of facs sorting:GFP+|time:Adult|replicate:Replicate 2,zfRNAadtP00R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Control|cell population of facs sorting:GFP+|time:Adult|replicate:Replicate 2,GSM4007786,GSM4007786: zfRNAadtP00R2; Danio rerio; RNA Seq,GSM4007786,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,Lds0P2_S4_R1_001.fastq.gz Lds0P2_S4_R2_001.fastq.gz,fastq fastq,8063674328.0,53050489.0,GSM4007786 r1,0:76 1:76,A:2142932036;C:1892279645;G:1965160014;T:2062649309;N:653324,76,76,,,2142932036,1892279645,1965160014,2062649309,653324,SRX6657757,SRS5218218,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.93858,0.94315,0.16694,0.16908,0.83782,0.84161,0.69345,0.73603,76,76,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53561,SRR9906799,SRX6657756,SRS5218217,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAadtP00R1,GSM4007785,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Control|cell population of facs sorting:GFP+|time:Adult|replicate:Replicate 1,zfRNAadtP00R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Control|cell population of facs sorting:GFP+|time:Adult|replicate:Replicate 1,GSM4007785,GSM4007785: zfRNAadtP00R1; Danio rerio; RNA Seq,GSM4007785,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,Lds0P1_S3_R1_001.fastq.gz Lds0P1_S3_R2_001.fastq.gz,fastq fastq,8088589256.0,53214403.0,GSM4007785 r1,0:76 1:76,A:2075935926;C:1970959110;G:2022264326;T:2018771586;N:658308,76,76,,,2075935926,1970959110,2022264326,2018771586,658308,SRX6657756,SRS5218217,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.92645,0.93309,0.17732,0.18266,0.84743,0.85121,0.72406,0.73766,76,76,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53562,SRR9906798,SRX6657755,SRS5218216,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldN36R2,GSM4007784,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP |time:36hr|replicate:Replicate 2,zfRNAldN36R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP |time:36hr|replicate:Replicate 2,GSM4007784,GSM4007784: zfRNAldN36R2; Danio rerio; RNA Seq,GSM4007784,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,TD36_2Neg_S8_R1_001.fastq.gz TD36_2Neg_S8_R2_001.fastq.gz,fastq fastq,5585507129.0,36977321.0,GSM4007784 r1,0:75.53 1:75.52,A:1441608710;C:1299899157;G:1382489435;T:1460670876;N:838951,75,75,,,1441608710,1299899157,1382489435,1460670876,838951,SRX6657755,SRS5218216,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.90161,0.90954,0.34163,0.36762,0.77208,0.77475,0.62098,0.62291,76,76,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53563,SRR9906797,SRX6657754,SRS5218215,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldN36R1,GSM4007783,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP |time:36hr|replicate:Replicate 1,zfRNAldN36R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP |time:36hr|replicate:Replicate 1,GSM4007783,GSM4007783: zfRNAldN36R1; Danio rerio; RNA Seq,GSM4007783,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,TD36_1Neg_S7_R1_001.fastq.gz TD36_1Neg_S7_R2_001.fastq.gz,fastq fastq,5251880856.0,34783967.0,GSM4007783 r1,0:75.50 1:75.48,A:1444102469;C:1132662042;G:1193758518;T:1480759618;N:598209,75,75,,,1444102469,1132662042,1193758518,1480759618,598209,SRX6657754,SRS5218215,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.8912,0.90553,0.35443,0.38081,0.76712,0.77212,0.53833,0.52968,76,76,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53564,SRR9906796,SRX6657753,SRS5218214,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldN00R2,GSM4007782,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Control|cell population of facs sorting:GFP |time:Adult|replicate:Replicate 2,zfRNAldN00R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Control|cell population of facs sorting:GFP |time:Adult|replicate:Replicate 2,GSM4007782,GSM4007782: zfRNAldN00R2; Danio rerio; RNA Seq,GSM4007782,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,TC2Neg_S12_R1_001.fastq.gz TC2Neg_S12_R2_001.fastq.gz,fastq fastq,5514036114.0,36521658.0,GSM4007782 r1,0:75.50 1:75.48,A:1534160858;C:1171528199;G:1254297458;T:1553209609;N:839990,75,75,,,1534160858,1171528199,1254297458,1553209609,839990,SRX6657753,SRS5218214,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.87319,0.89652,0.42952,0.45836,0.76822,0.7725,0.55467,0.54896,74,76,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53565,SRR9906795,SRX6657752,SRS5218213,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldN00R1,GSM4007781,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Control|cell population of facs sorting:GFP |time:Adult|replicate:Replicate 1,zfRNAldN00R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Control|cell population of facs sorting:GFP |time:Adult|replicate:Replicate 1,GSM4007781,GSM4007781: zfRNAldN00R1; Danio rerio; RNA Seq,GSM4007781,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,TC1Neg_S11_R1_001.fastq.gz TC1Neg_S11_R2_001.fastq.gz,fastq fastq,6356582607.0,42101680.0,GSM4007781 r1,0:75.50 1:75.49,A:1786373439;C:1330051743;G:1429422994;T:1809862022;N:872409,75,75,,,1786373439,1330051743,1429422994,1809862022,872409,SRX6657752,SRS5218213,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.86968,0.89454,0.42965,0.45666,0.76684,0.76954,0.53875,0.5273,76,76,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53566,SRR9906794,SRX6657751,SRS5218212,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldP36R2,GSM4007780,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:36hr|replicate:Replicate 2,zfRNAldP36R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:36hr|replicate:Replicate 2,GSM4007780,GSM4007780: zfRNAldP36R2; Danio rerio; RNA Seq,GSM4007780,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,TD36_2Pos_S6_R1_001.fastq.gz TD36_2Pos_S6_R2_001.fastq.gz,fastq fastq,4538979938.0,30037021.0,GSM4007780 r1,0:75.56 1:75.56,A:1121835185;C:1107349762;G:1178657723;T:1130551790;N:585478,75,75,,,1121835185,1107349762,1178657723,1130551790,585478,SRX6657751,SRS5218212,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.92939,0.93856,0.26301,0.28237,0.78127,0.78484,0.63081,0.64308,75,76,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53567,SRR9906793,SRX6657750,SRS5218211,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldP36R1,GSM4007779,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:36hr|replicate:Replicate 1,zfRNAldP36R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Light damage|cell population of facs sorting:GFP+|time:36hr|replicate:Replicate 1,GSM4007779,GSM4007779: zfRNAldP36R1; Danio rerio; RNA Seq,GSM4007779,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,TD36_1Pos_S5_R1_001.fastq.gz TD36_1Pos_S5_R2_001.fastq.gz,fastq fastq,4731594110.0,31316303.0,GSM4007779 r1,0:75.55 1:75.54,A:1185964667;C:1138861246;G:1209345019;T:1196771621;N:651557,75,75,,,1185964667,1138861246,1209345019,1196771621,651557,SRX6657750,SRS5218211,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.9246,0.93426,0.26174,0.28336,0.77479,0.77784,0.60646,0.60843,74,76,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53568,SRR9906792,SRX6657749,SRS5218210,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldP00R2,GSM4007778,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Control|cell population of facs sorting:GFP+|time:Adult|replicate:Replicate 2,zfRNAldP00R2,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Control|cell population of facs sorting:GFP+|time:Adult|replicate:Replicate 2,GSM4007778,GSM4007778: zfRNAldP00R2; Danio rerio; RNA Seq,GSM4007778,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,TC2Pos_S10_R1_001.fastq.gz TC2Pos_S10_R2_001.fastq.gz,fastq fastq,5591137280.0,37029316.0,GSM4007778 r1,0:75.50 1:75.49,A:1526678021;C:1222716619;G:1298242890;T:1542649889;N:849861,75,75,,,1526678021,1222716619,1298242890,1542649889,849861,SRX6657749,SRS5218210,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.88181,0.90227,0.37932,0.4036,0.79768,0.79989,0.56364,0.5577,76,76,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 53569,SRR9906791,SRX6657748,SRS5218209,SRP217505,PRJNA558770,Comparative transcriptomic and epigenomic analysis identifies key regulators of injury response and neurogenic competence in retinal glia,GSE135406,Other,Injury induces retinal Muller glia of non mammalian but not mammalian vertebrates to generate neurons. To identify gene regulatory networks that control neurogenic competence in retinal glia we used bulk and single cell RNA seq and ATAC seq analysis to comprehensively profile gene expression and chromatin conformation in Muller glia from zebrafish chick and mice. This was conducted during glial development following inner and outer retinal injury as well as following treatment with extrinsic factors that induce glial reprogramming. Integration of these data together with functional analysis of candidate genes identified evolutionarily conserved and species specific gene regulatory networks controlling glial quiescence gliosis and neurogenic competence. In zebrafish and chick transition from quiescence to gliosis is a necessary stage in acquisition of neurogenic competence while in mice a dedicated network suppresses this transition and rapidly restores quiescence. These findings may help guide the design of cell based therapies aimed at restoring retinal neurons lost to disease. Overall design: In this study to comprehensively identify transcriptional and epigenetic regulators of neurogenic competence in MG we profiled mRNA levels using bulk RNA seq and chromatin accessibility using ATAC Assay for Transposase Accessible Chromatin sequencing technology in zebrafish and mouse in response to multiple neuronal injury models as well as growth factor treatment. In total we generated 105 bulk RNA Seq libraries including 5 technical replicates and 40 bulk ATAC Seq libraries.,,pubmed:33004674;pubmed:35259089;pubmed:38123561,,zfRNAldP00R1,GSM4007777,,source name:Retina|tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Control|cell population of facs sorting:GFP+|time:Adult|replicate:Replicate 1,zfRNAldP00R1,Raw data from mouse and zebrafish were separately mapped to the GRCm38/mm10 and GRCz10/danRer10 genome assembly using STAR Raw counts of genes were further used to calculate FPKM Fragments Per Kilobase Of Exon Per Million and identify differentially expressed genes through EdgeR post removing adaptors using cutadapt 58 50bp paired end ATAC Seq reads from zebrafish and mouse were separately aligned to GRCz10/danRer10 and GRCm38/mm10 reference genome using Bowtie2 with default parameters We filtered reads from chromosome M and Y and included high mapping quality reads MAPQ score > 10 through SAMTools for further analysis. Duplicate reads was removed using Picard tools MarkDuplicates program. ATAC Seq peak regions were called using MACS2 with parameters nomodel shift 100 extsize 200. The blacklisted regions in mouse were excluded from peak regions https://www.encodeproject.org/annotations/ENCSR636HFF/. We counted the raw fragments for each peak region using HTSeq. Genome build: GRCm38/mm10 and GRCz10/danRer10 Supplementary files format and content: FPKM files for gene expression. Peak files in narrowPeak format with following columns: chromosome start stop name length of peak region strand integer score for display fold change log10pvalue log10qvalue relative summit position to peak start.,Retina,For NMDA damage adult mice either CD1 or GLASTCreERT2; Sun1 sGFP mice at 2 month of age were anesthetized with isoflurane inhalation. A puncture was made just behind the limbus with a 30G needle. Two microliters of 100mM NMDA in PBS was intravitreally injected using a syringe with a 33G blunt ended needle. Mice were sacrificed and retinas were collected at indicated timepoints. For light damage the mice were reared in cyclic 12 hour low light/12 hour dark conditions at the University of Florida animal housing facility. Prior to light damage mice were placed in a modified cage equipped with dimmable white light LED strips. Light intensity was measured using a light meter Thermo Fisher Scientific Inc. Waltham MA and set to 2000 lux. To induce death of rods and cones adult albino; Tg[gfap:EGFP]nt11 fish were dark adapted for 14 days then transferred to clear polycarbonate tanks placed between four fluorescent bulbs 20 000 lux and water temperature maintained at 32°C for up to 72 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water. To induce amacrine and ganglion cell death we injected N methyl D aspartic acid NMDA Sigma Aldrich; St. Louis MO intravitreally. Adult Tg[gfap:GFP]mi2001 zebrafish were anesthetized in 0.1% 2 phenoxyethanol. A sapphire blade was used to make an incision in the posterior cornea and 0.5 μl of 100 mM NMDA was injected into the intravitreal space of the eye using a 33 gauge Hamilton syringe into the intravitreal space of the eye. The fish were revived and placed in a 32°C dark incubator for up to 36 hours. Fish were euthanized by anesthetic overdose of 0.2% 2 phenoxyethanol in system water.,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,To induce Cre recombination GLASTCreERT2; Sun1 sGFP mice at 2 weeks of age were intraperitoneally injected with Tamoxifen in corn oil for xxx consecutive days at xxxmg/dose. Nfialox/lox; Nfiblox/lox and Nfixlox/lox mice were crossed to GLASTCreERT2; Sun1 sGFP mice. To generate MG specific loss of function mutants of NFIa/b/x genes 3 wpf GLASTCreERT2; NFIa/bxlox/lox;Sun1 sGFP mice were fed with tamoxifen diet for 3 weeks following by 2 weeks with normal diet. All mice were housed in a climate controlled pathogen free facility on a 14 h 10 h light/dark cycle 07:00 lights on – 19:00 lights off. The adult zebrafish used in these studies were 6 mpf to 12 mpf 3 5cm in length. Fish were maintained under a light and dark cycle of 14 hours light and 10 hours of dark at 28.5°C.,tissue:Retina|genotype:Tg[gfap:EGFP]nt11 albino|condition:Control|cell population of facs sorting:GFP+|time:Adult|replicate:Replicate 1,GSM4007777,GSM4007777: zfRNAldP00R1; Danio rerio; RNA Seq,GSM4007777,,1,RNA was extracted from both GFP positive and GFP negative cell fractions using miRNAeasy Mini Kit #217004 Qiagen. Ribosomal RNA was depleted and total RNA was captured from the RNA samples using Illumina TruSeq Stranded RNA LT kit Ribo ZeroTM Gold # 15032619 Illumina. For tagmentation cell nuclei were incubated with 2.5 UL enzyme in 50UL total volume at 37°C in a thermocycler Illumnia Nextera DNA library prep kit #FC1211030. DNA was cleaned up using MinElute PCR purification kit #28006 Qiagen and eluted in 10UL of EB buffer. Flow sorted RNA samples were sent to the Deep Sequencing and Microarray Core Johns Hopkins University for library preparation and sequencing. Around 8 10 libraries were pooled and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run resulting in 45 55 million reads per library. Sorted cells 50k 75k from GFP positive samples were used for ATAC library preparation. Completed ATAC Seq libraries were then analyzed by Fragment Bioanalyzer and sequenced for paired end 75 cycles using the NextSeq 500 system with 400 500 million reads per run.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP217505,,,TC1Pos_S4_R1_001.fastq.gz TC1Pos_S4_R2_001.fastq.gz,fastq fastq,5517591671.0,36535124.0,GSM4007777 r1,0:75.52 1:75.50,A:1482487831;C:1223340071;G:1303339516;T:1507615853;N:808400,75,75,,,1482487831,1223340071,1303339516,1507615853,808400,SRX6657748,SRS5218209,SRA934563,GEO,"Ophthalmology, Johns Hopkins University",2,0.88954,0.90541,0.36969,0.39394,0.79261,0.79299,0.56093,0.576,76,76,B,B,biological fallback assumption,illumina,nextseq,unknown,rrna_depletion,trueseq,sc_generic,bulk,bulk,,United States,2019-08-05,Multi-stage,Multi-stage,Eye,Sensory System 55210,SRR10153634,SRX6879049,SRS5414797,SRP222804,PRJNA573071,Bulk transcriptomic analysis of two models of zebrafish beta catenin driven HCC,GSE137787,Transcriptome Analysis,Up to 41% of hepatocellular carcinomas HCCs result from activating mutations in the CTNNB1 gene encoding ß catenin. ß catenin has dual cellular functions as a component of the Wnt signaling pathway and adherens junctions. HCC associated CTNNB1 mutations stabilize the ß catenin protein leading to nuclear and/or cytoplasmic localization of ß catenin and downstream activation of Wnt target genes. In patient HCC samples ß catenin nuclear and cytoplasmic localization are typically patchy even among HCC with highly active CTNNB1 mutations. The functional and clinical relevance of this heterogeneity in ß catenin activation are not well understood. To define mechanisms of ß catenin driven HCC initiation we generated a Cre lox system that enabled switching on activated ß catenin in 1 a small number of hepatocytes in early development; or 2 the majority of hepatocytes in later development or maturity. We discovered that switching on activated ß catenin in a subset of larval hepatocytes was sufficient to drive HCC initiation. To determine the role of Wnt/ß catenin signaling heterogeneity later in hepatocarcinogenesis we performed RNA seq analysis of zebrafish ß catenin driven HCC. Ingenuity Pathway Analysis of differentially expressed genes in the Cre lox HCC model revealed that “Cancer” and “Liver Tumor” categories were significantly altered indicating transcriptional similarities with human HCC and other vertebrate HCC models. At the single cell level 2.9% to 15.2% of hepatocytes from zebrafish ß catenin driven HCC expressed two or more of the Wnt target genes axin2 mtor glula myca and wif1 indicating focal activation of Wnt signaling in established tumors. Thus heterogeneous ß catenin activation drives HCC initiation and persists throughout hepatocarcinogenesis. Overall design: Examination of HCC and nonHCC liver samples from two different models of zebrafish compared to wildtype/nonHCC siblings.,parent bioproject:PRJNA573068,pubmed:31575545,,609,GSM4087869,,tissue:Liver|transgenic line:Tgfabp10a CreERT2;fabp10a flox beta catenin|age:11 mpf at larval stg:10micromolar 4 hydroxytamoxifen from 3 dpf to 6 dpf HCC,609,The Zebrafish GRCz11 FASTA and GTF files were downloaded from Ensembl release 94. The reference database was created using STAR version 2.6.1b with splice junctions optimized for 125 base pair reads.  Reads were trimmed of adapters and aligned to the reference database using STAR in two pass mode to output a BAM file sorted by coordinates.  Mapped reads were assigned to annotated genes in the GTF file using featureCounts version 1.6.3.  Differentially expressed genes were identified using a 5% false discovery rate with DESeq2 version 1.22.2. Genome build: GRCz11 Supplementary files format and content: Raw gene expression quantification with featureCounts v1.6.3 was collated into a tab delimited matrix where each column denotes a sample and each row corresponds to a gene.,Liver,Larvae were treated with either 0.1% ethanol or 10micromolar 4 hydroxytamoxifen from 3 dpf to 6 dpf,Zebrafish were euthanized by rapid chilling and their livers were dissected. Half of each liver was submitted for histologic evaluation to confirm the diagnosis HCC or no HCC. The other half of each dissected liver was flash frozen on dry ice and stored in 80C prior to RNA extraction. RNA was isolated using TriReagent Cat# R2050 1 50 Zymo Research and Direct Zol RNA Mini prep kit Cat# R2050 Zymo Research including DNase treatment following manufacturer’s protocols. Samples were eluted in RNase /DNase free water. Sample quality control library preparation sequencing and alignments were performed by the Huntsman Cancer Institute HCI High Throughput Genomics and Bioinformatic Analysis Shared Resource. Paired read 150 base pair sequencing was performed using Illumina’s NovaSeq instrument. Three or four pooled libraries were run on each lane of the flow cell. Illumina TruSeq Stranded mRNA Library Prep RIN 8 10,Fish were raised following IACUC approved protocols in insititutional zebrafish facility.,transgenic line:Tgfabp10a CreERT2;fabp10a flox beta catenin|age:11 mpf at larval stg:10micromolar 4 hydroxytamoxifen from 3 dpf to 6 dpf HCC,GSM4087869,GSM4087869: 609; Danio rerio; RNA Seq,GSM4087869,,1,Zebrafish were euthanized by rapid chilling and their livers were dissected. Half of each liver was submitted for histologic evaluation to confirm the diagnosis HCC or no HCC. The other half of each dissected liver was flash frozen on dry ice and stored in 80C prior to RNA extraction. RNA was isolated using TriReagent Cat# R2050 1 50 Zymo Research and Direct Zol RNA Mini prep kit Cat# R2050 Zymo Research including DNase treatment following manufacturer's protocols. Samples were eluted in RNase /DNase free water. Sample quality control library preparation sequencing and alignments were performed by the Huntsman Cancer Institute HCI High Throughput Genomics and Bioinformatic Analysis Shared Resource. Paired read 150 base pair sequencing was performed using Illumina's NovaSeq instrument. Three or four pooled libraries were run on each lane of the flow cell. Illumina TruSeq Stranded mRNA Library Prep RIN 8 10,GEO Accession:GSM4087869,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP222804,,,16048X9_190607_A00421_0071_BHL22MDSXX_S20_L004_R1_001.fastq.gz 16048X9_190607_A00421_0071_BHL22MDSXX_S20_L004_R2_001.fastq.gz,fastq fastq,9109094026.0,30162563.0,GSM4087869 r1,0:151 1:151,A:2407747194;C:2151727590;G:2238603631;T:2310854013;N:161598,151,151,,,2407747194,2151727590,2238603631,2310854013,161598,SRX6879049,SRS5414797,SRA965568,GEO,"Pathology, University of California, San Francisco",2,0.95438,0.95659,0.04172,0.04068,0.80267,0.80308,0.56788,0.56779,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,United States,2019-09-20,Multi-stage,Multi-stage,Liver,Liver and Biliary System 55211,SRR10153633,SRX6879048,SRS5414796,SRP222804,PRJNA573071,Bulk transcriptomic analysis of two models of zebrafish beta catenin driven HCC,GSE137787,Transcriptome Analysis,Up to 41% of hepatocellular carcinomas HCCs result from activating mutations in the CTNNB1 gene encoding ß catenin. ß catenin has dual cellular functions as a component of the Wnt signaling pathway and adherens junctions. HCC associated CTNNB1 mutations stabilize the ß catenin protein leading to nuclear and/or cytoplasmic localization of ß catenin and downstream activation of Wnt target genes. In patient HCC samples ß catenin nuclear and cytoplasmic localization are typically patchy even among HCC with highly active CTNNB1 mutations. The functional and clinical relevance of this heterogeneity in ß catenin activation are not well understood. To define mechanisms of ß catenin driven HCC initiation we generated a Cre lox system that enabled switching on activated ß catenin in 1 a small number of hepatocytes in early development; or 2 the majority of hepatocytes in later development or maturity. We discovered that switching on activated ß catenin in a subset of larval hepatocytes was sufficient to drive HCC initiation. To determine the role of Wnt/ß catenin signaling heterogeneity later in hepatocarcinogenesis we performed RNA seq analysis of zebrafish ß catenin driven HCC. Ingenuity Pathway Analysis of differentially expressed genes in the Cre lox HCC model revealed that “Cancer” and “Liver Tumor” categories were significantly altered indicating transcriptional similarities with human HCC and other vertebrate HCC models. At the single cell level 2.9% to 15.2% of hepatocytes from zebrafish ß catenin driven HCC expressed two or more of the Wnt target genes axin2 mtor glula myca and wif1 indicating focal activation of Wnt signaling in established tumors. Thus heterogeneous ß catenin activation drives HCC initiation and persists throughout hepatocarcinogenesis. Overall design: Examination of HCC and nonHCC liver samples from two different models of zebrafish compared to wildtype/nonHCC siblings.,parent bioproject:PRJNA573068,pubmed:31575545,,624,GSM4087868,,tissue:Liver|transgenic line:Tgfabp10a CreERT2;fabp10a flox beta catenin|age:11 mpf at larval stg:10micromolar 4 hydroxytamoxifen from 3 dpf to 6 dpf,624,The Zebrafish GRCz11 FASTA and GTF files were downloaded from Ensembl release 94. The reference database was created using STAR version 2.6.1b with splice junctions optimized for 125 base pair reads.  Reads were trimmed of adapters and aligned to the reference database using STAR in two pass mode to output a BAM file sorted by coordinates.  Mapped reads were assigned to annotated genes in the GTF file using featureCounts version 1.6.3.  Differentially expressed genes were identified using a 5% false discovery rate with DESeq2 version 1.22.2. Genome build: GRCz11 Supplementary files format and content: Raw gene expression quantification with featureCounts v1.6.3 was collated into a tab delimited matrix where each column denotes a sample and each row corresponds to a gene.,Liver,Larvae were treated with either 0.1% ethanol or 10micromolar 4 hydroxytamoxifen from 3 dpf to 6 dpf,Zebrafish were euthanized by rapid chilling and their livers were dissected. Half of each liver was submitted for histologic evaluation to confirm the diagnosis HCC or no HCC. The other half of each dissected liver was flash frozen on dry ice and stored in 80C prior to RNA extraction. RNA was isolated using TriReagent Cat# R2050 1 50 Zymo Research and Direct Zol RNA Mini prep kit Cat# R2050 Zymo Research including DNase treatment following manufacturer’s protocols. Samples were eluted in RNase /DNase free water. Sample quality control library preparation sequencing and alignments were performed by the Huntsman Cancer Institute HCI High Throughput Genomics and Bioinformatic Analysis Shared Resource. Paired read 150 base pair sequencing was performed using Illumina’s NovaSeq instrument. Three or four pooled libraries were run on each lane of the flow cell. Illumina TruSeq Stranded mRNA Library Prep RIN 8 10,Fish were raised following IACUC approved protocols in insititutional zebrafish facility.,transgenic line:Tgfabp10a CreERT2;fabp10a flox beta catenin|age:11 mpf at larval stg:10micromolar 4 hydroxytamoxifen from 3 dpf to 6 dpf,GSM4087868,GSM4087868: 624; Danio rerio; RNA Seq,GSM4087868,,1,Zebrafish were euthanized by rapid chilling and their livers were dissected. Half of each liver was submitted for histologic evaluation to confirm the diagnosis HCC or no HCC. The other half of each dissected liver was flash frozen on dry ice and stored in 80C prior to RNA extraction. RNA was isolated using TriReagent Cat# R2050 1 50 Zymo Research and Direct Zol RNA Mini prep kit Cat# R2050 Zymo Research including DNase treatment following manufacturer's protocols. Samples were eluted in RNase /DNase free water. Sample quality control library preparation sequencing and alignments were performed by the Huntsman Cancer Institute HCI High Throughput Genomics and Bioinformatic Analysis Shared Resource. Paired read 150 base pair sequencing was performed using Illumina's NovaSeq instrument. Three or four pooled libraries were run on each lane of the flow cell. Illumina TruSeq Stranded mRNA Library Prep RIN 8 10,GEO Accession:GSM4087868,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP222804,,,16048X8_190607_A00421_0071_BHL22MDSXX_S19_L004_R1_001.fastq.gz 16048X8_190607_A00421_0071_BHL22MDSXX_S19_L004_R2_001.fastq.gz,fastq fastq,9818272170.0,32510835.0,GSM4087868 r1,0:151 1:151,A:2609919214;C:2318388621;G:2406490919;T:2483290917;N:182499,151,151,,,2609919214,2318388621,2406490919,2483290917,182499,SRX6879048,SRS5414796,SRA965568,GEO,"Pathology, University of California, San Francisco",2,0.95073,0.95151,0.04785,0.0469,0.80919,0.81055,0.54958,0.55772,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,United States,2019-09-20,Multi-stage,Multi-stage,Liver,Liver and Biliary System 55213,SRR10153631,SRX6879046,SRS5414794,SRP222804,PRJNA573071,Bulk transcriptomic analysis of two models of zebrafish beta catenin driven HCC,GSE137787,Transcriptome Analysis,Up to 41% of hepatocellular carcinomas HCCs result from activating mutations in the CTNNB1 gene encoding ß catenin. ß catenin has dual cellular functions as a component of the Wnt signaling pathway and adherens junctions. HCC associated CTNNB1 mutations stabilize the ß catenin protein leading to nuclear and/or cytoplasmic localization of ß catenin and downstream activation of Wnt target genes. In patient HCC samples ß catenin nuclear and cytoplasmic localization are typically patchy even among HCC with highly active CTNNB1 mutations. The functional and clinical relevance of this heterogeneity in ß catenin activation are not well understood. To define mechanisms of ß catenin driven HCC initiation we generated a Cre lox system that enabled switching on activated ß catenin in 1 a small number of hepatocytes in early development; or 2 the majority of hepatocytes in later development or maturity. We discovered that switching on activated ß catenin in a subset of larval hepatocytes was sufficient to drive HCC initiation. To determine the role of Wnt/ß catenin signaling heterogeneity later in hepatocarcinogenesis we performed RNA seq analysis of zebrafish ß catenin driven HCC. Ingenuity Pathway Analysis of differentially expressed genes in the Cre lox HCC model revealed that “Cancer” and “Liver Tumor” categories were significantly altered indicating transcriptional similarities with human HCC and other vertebrate HCC models. At the single cell level 2.9% to 15.2% of hepatocytes from zebrafish ß catenin driven HCC expressed two or more of the Wnt target genes axin2 mtor glula myca and wif1 indicating focal activation of Wnt signaling in established tumors. Thus heterogeneous ß catenin activation drives HCC initiation and persists throughout hepatocarcinogenesis. Overall design: Examination of HCC and nonHCC liver samples from two different models of zebrafish compared to wildtype/nonHCC siblings.,parent bioproject:PRJNA573068,pubmed:31575545,,625,GSM4087866,,tissue:Liver|transgenic line:Tgfabp10a CreERT2;fabp10a flox beta catenin|age:11 mpf at larval stg:10micromolar 4 hydroxytamoxifen from 3 dpf to 6 dpf HCC,625,The Zebrafish GRCz11 FASTA and GTF files were downloaded from Ensembl release 94. The reference database was created using STAR version 2.6.1b with splice junctions optimized for 125 base pair reads.  Reads were trimmed of adapters and aligned to the reference database using STAR in two pass mode to output a BAM file sorted by coordinates.  Mapped reads were assigned to annotated genes in the GTF file using featureCounts version 1.6.3.  Differentially expressed genes were identified using a 5% false discovery rate with DESeq2 version 1.22.2. Genome build: GRCz11 Supplementary files format and content: Raw gene expression quantification with featureCounts v1.6.3 was collated into a tab delimited matrix where each column denotes a sample and each row corresponds to a gene.,Liver,Larvae were treated with either 0.1% ethanol or 10micromolar 4 hydroxytamoxifen from 3 dpf to 6 dpf,Zebrafish were euthanized by rapid chilling and their livers were dissected. Half of each liver was submitted for histologic evaluation to confirm the diagnosis HCC or no HCC. The other half of each dissected liver was flash frozen on dry ice and stored in 80C prior to RNA extraction. RNA was isolated using TriReagent Cat# R2050 1 50 Zymo Research and Direct Zol RNA Mini prep kit Cat# R2050 Zymo Research including DNase treatment following manufacturer’s protocols. Samples were eluted in RNase /DNase free water. Sample quality control library preparation sequencing and alignments were performed by the Huntsman Cancer Institute HCI High Throughput Genomics and Bioinformatic Analysis Shared Resource. Paired read 150 base pair sequencing was performed using Illumina’s NovaSeq instrument. Three or four pooled libraries were run on each lane of the flow cell. Illumina TruSeq Stranded mRNA Library Prep RIN 8 10,Fish were raised following IACUC approved protocols in insititutional zebrafish facility.,transgenic line:Tgfabp10a CreERT2;fabp10a flox beta catenin|age:11 mpf at larval stg:10micromolar 4 hydroxytamoxifen from 3 dpf to 6 dpf HCC,GSM4087866,GSM4087866: 625; Danio rerio; RNA Seq,GSM4087866,,1,Zebrafish were euthanized by rapid chilling and their livers were dissected. Half of each liver was submitted for histologic evaluation to confirm the diagnosis HCC or no HCC. The other half of each dissected liver was flash frozen on dry ice and stored in 80C prior to RNA extraction. RNA was isolated using TriReagent Cat# R2050 1 50 Zymo Research and Direct Zol RNA Mini prep kit Cat# R2050 Zymo Research including DNase treatment following manufacturer's protocols. Samples were eluted in RNase /DNase free water. Sample quality control library preparation sequencing and alignments were performed by the Huntsman Cancer Institute HCI High Throughput Genomics and Bioinformatic Analysis Shared Resource. Paired read 150 base pair sequencing was performed using Illumina's NovaSeq instrument. Three or four pooled libraries were run on each lane of the flow cell. Illumina TruSeq Stranded mRNA Library Prep RIN 8 10,GEO Accession:GSM4087866,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP222804,,,16048X6_190607_A00421_0071_BHL22MDSXX_S17_L004_R2_001.fastq.gz 16048X6_190607_A00421_0071_BHL22MDSXX_S17_L004_R1_001.fastq.gz,fastq fastq,9650060284.0,31953842.0,GSM4087866 r1,0:151 1:151,A:2558021442;C:2277296580;G:2371770140;T:2442792643;N:179479,151,151,,,2558021442,2277296580,2371770140,2442792643,179479,SRX6879046,SRS5414794,SRA965568,GEO,"Pathology, University of California, San Francisco",2,0.95395,0.95537,0.0475,0.04582,0.79947,0.80046,0.53471,0.54384,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,United States,2019-09-20,Multi-stage,Multi-stage,Liver,Liver and Biliary System 55214,SRR10153630,SRX6879045,SRS5414793,SRP222804,PRJNA573071,Bulk transcriptomic analysis of two models of zebrafish beta catenin driven HCC,GSE137787,Transcriptome Analysis,Up to 41% of hepatocellular carcinomas HCCs result from activating mutations in the CTNNB1 gene encoding ß catenin. ß catenin has dual cellular functions as a component of the Wnt signaling pathway and adherens junctions. HCC associated CTNNB1 mutations stabilize the ß catenin protein leading to nuclear and/or cytoplasmic localization of ß catenin and downstream activation of Wnt target genes. In patient HCC samples ß catenin nuclear and cytoplasmic localization are typically patchy even among HCC with highly active CTNNB1 mutations. The functional and clinical relevance of this heterogeneity in ß catenin activation are not well understood. To define mechanisms of ß catenin driven HCC initiation we generated a Cre lox system that enabled switching on activated ß catenin in 1 a small number of hepatocytes in early development; or 2 the majority of hepatocytes in later development or maturity. We discovered that switching on activated ß catenin in a subset of larval hepatocytes was sufficient to drive HCC initiation. To determine the role of Wnt/ß catenin signaling heterogeneity later in hepatocarcinogenesis we performed RNA seq analysis of zebrafish ß catenin driven HCC. Ingenuity Pathway Analysis of differentially expressed genes in the Cre lox HCC model revealed that “Cancer” and “Liver Tumor” categories were significantly altered indicating transcriptional similarities with human HCC and other vertebrate HCC models. At the single cell level 2.9% to 15.2% of hepatocytes from zebrafish ß catenin driven HCC expressed two or more of the Wnt target genes axin2 mtor glula myca and wif1 indicating focal activation of Wnt signaling in established tumors. Thus heterogeneous ß catenin activation drives HCC initiation and persists throughout hepatocarcinogenesis. Overall design: Examination of HCC and nonHCC liver samples from two different models of zebrafish compared to wildtype/nonHCC siblings.,parent bioproject:PRJNA573068,pubmed:31575545,,623,GSM4087865,,tissue:Liver|transgenic line:Tgfabp10a CreERT2;fabp10a flox beta catenin|age:11 mpf at larval stg:10micromolar 4 hydroxytamoxifen from 3 dpf to 6 dpf,623,The Zebrafish GRCz11 FASTA and GTF files were downloaded from Ensembl release 94. The reference database was created using STAR version 2.6.1b with splice junctions optimized for 125 base pair reads.  Reads were trimmed of adapters and aligned to the reference database using STAR in two pass mode to output a BAM file sorted by coordinates.  Mapped reads were assigned to annotated genes in the GTF file using featureCounts version 1.6.3.  Differentially expressed genes were identified using a 5% false discovery rate with DESeq2 version 1.22.2. Genome build: GRCz11 Supplementary files format and content: Raw gene expression quantification with featureCounts v1.6.3 was collated into a tab delimited matrix where each column denotes a sample and each row corresponds to a gene.,Liver,Larvae were treated with either 0.1% ethanol or 10micromolar 4 hydroxytamoxifen from 3 dpf to 6 dpf,Zebrafish were euthanized by rapid chilling and their livers were dissected. Half of each liver was submitted for histologic evaluation to confirm the diagnosis HCC or no HCC. The other half of each dissected liver was flash frozen on dry ice and stored in 80C prior to RNA extraction. RNA was isolated using TriReagent Cat# R2050 1 50 Zymo Research and Direct Zol RNA Mini prep kit Cat# R2050 Zymo Research including DNase treatment following manufacturer’s protocols. Samples were eluted in RNase /DNase free water. Sample quality control library preparation sequencing and alignments were performed by the Huntsman Cancer Institute HCI High Throughput Genomics and Bioinformatic Analysis Shared Resource. Paired read 150 base pair sequencing was performed using Illumina’s NovaSeq instrument. Three or four pooled libraries were run on each lane of the flow cell. Illumina TruSeq Stranded mRNA Library Prep RIN 8 10,Fish were raised following IACUC approved protocols in insititutional zebrafish facility.,transgenic line:Tgfabp10a CreERT2;fabp10a flox beta catenin|age:11 mpf at larval stg:10micromolar 4 hydroxytamoxifen from 3 dpf to 6 dpf,GSM4087865,GSM4087865: 623; Danio rerio; RNA Seq,GSM4087865,,1,Zebrafish were euthanized by rapid chilling and their livers were dissected. Half of each liver was submitted for histologic evaluation to confirm the diagnosis HCC or no HCC. The other half of each dissected liver was flash frozen on dry ice and stored in 80C prior to RNA extraction. RNA was isolated using TriReagent Cat# R2050 1 50 Zymo Research and Direct Zol RNA Mini prep kit Cat# R2050 Zymo Research including DNase treatment following manufacturer's protocols. Samples were eluted in RNase /DNase free water. Sample quality control library preparation sequencing and alignments were performed by the Huntsman Cancer Institute HCI High Throughput Genomics and Bioinformatic Analysis Shared Resource. Paired read 150 base pair sequencing was performed using Illumina's NovaSeq instrument. Three or four pooled libraries were run on each lane of the flow cell. Illumina TruSeq Stranded mRNA Library Prep RIN 8 10,GEO Accession:GSM4087865,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP222804,,,16048X5_190607_A00421_0071_BHL22MDSXX_S16_L004_R2_001.fastq.gz 16048X5_190607_A00421_0071_BHL22MDSXX_S16_L004_R1_001.fastq.gz,fastq fastq,11046705188.0,36578494.0,GSM4087865 r1,0:151 1:151,A:2912202858;C:2634649411;G:2724125067;T:2775523373;N:204479,151,151,,,2912202858,2634649411,2724125067,2775523373,204479,SRX6879045,SRS5414793,SRA965568,GEO,"Pathology, University of California, San Francisco",2,0.95836,0.95975,0.04299,0.04253,0.79433,0.795,0.55057,0.55465,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,United States,2019-09-20,Multi-stage,Multi-stage,Liver,Liver and Biliary System 55217,SRR10153627,SRX6879042,SRS5414790,SRP222804,PRJNA573071,Bulk transcriptomic analysis of two models of zebrafish beta catenin driven HCC,GSE137787,Transcriptome Analysis,Up to 41% of hepatocellular carcinomas HCCs result from activating mutations in the CTNNB1 gene encoding ß catenin. ß catenin has dual cellular functions as a component of the Wnt signaling pathway and adherens junctions. HCC associated CTNNB1 mutations stabilize the ß catenin protein leading to nuclear and/or cytoplasmic localization of ß catenin and downstream activation of Wnt target genes. In patient HCC samples ß catenin nuclear and cytoplasmic localization are typically patchy even among HCC with highly active CTNNB1 mutations. The functional and clinical relevance of this heterogeneity in ß catenin activation are not well understood. To define mechanisms of ß catenin driven HCC initiation we generated a Cre lox system that enabled switching on activated ß catenin in 1 a small number of hepatocytes in early development; or 2 the majority of hepatocytes in later development or maturity. We discovered that switching on activated ß catenin in a subset of larval hepatocytes was sufficient to drive HCC initiation. To determine the role of Wnt/ß catenin signaling heterogeneity later in hepatocarcinogenesis we performed RNA seq analysis of zebrafish ß catenin driven HCC. Ingenuity Pathway Analysis of differentially expressed genes in the Cre lox HCC model revealed that “Cancer” and “Liver Tumor” categories were significantly altered indicating transcriptional similarities with human HCC and other vertebrate HCC models. At the single cell level 2.9% to 15.2% of hepatocytes from zebrafish ß catenin driven HCC expressed two or more of the Wnt target genes axin2 mtor glula myca and wif1 indicating focal activation of Wnt signaling in established tumors. Thus heterogeneous ß catenin activation drives HCC initiation and persists throughout hepatocarcinogenesis. Overall design: Examination of HCC and nonHCC liver samples from two different models of zebrafish compared to wildtype/nonHCC siblings.,parent bioproject:PRJNA573068,pubmed:31575545,,605,GSM4087862,,tissue:Liver|transgenic line:Tgfabp10a CreERT2;fabp10a flox beta catenin|age:11 mpf at larval stg:10micromolar 4 hydroxytamoxifen from 3 dpf to 6 dpf,605,The Zebrafish GRCz11 FASTA and GTF files were downloaded from Ensembl release 94. The reference database was created using STAR version 2.6.1b with splice junctions optimized for 125 base pair reads.  Reads were trimmed of adapters and aligned to the reference database using STAR in two pass mode to output a BAM file sorted by coordinates.  Mapped reads were assigned to annotated genes in the GTF file using featureCounts version 1.6.3.  Differentially expressed genes were identified using a 5% false discovery rate with DESeq2 version 1.22.2. Genome build: GRCz11 Supplementary files format and content: Raw gene expression quantification with featureCounts v1.6.3 was collated into a tab delimited matrix where each column denotes a sample and each row corresponds to a gene.,Liver,Larvae were treated with either 0.1% ethanol or 10micromolar 4 hydroxytamoxifen from 3 dpf to 6 dpf,Zebrafish were euthanized by rapid chilling and their livers were dissected. Half of each liver was submitted for histologic evaluation to confirm the diagnosis HCC or no HCC. The other half of each dissected liver was flash frozen on dry ice and stored in 80C prior to RNA extraction. RNA was isolated using TriReagent Cat# R2050 1 50 Zymo Research and Direct Zol RNA Mini prep kit Cat# R2050 Zymo Research including DNase treatment following manufacturer’s protocols. Samples were eluted in RNase /DNase free water. Sample quality control library preparation sequencing and alignments were performed by the Huntsman Cancer Institute HCI High Throughput Genomics and Bioinformatic Analysis Shared Resource. Paired read 150 base pair sequencing was performed using Illumina’s NovaSeq instrument. Three or four pooled libraries were run on each lane of the flow cell. Illumina TruSeq Stranded mRNA Library Prep RIN 8 10,Fish were raised following IACUC approved protocols in insititutional zebrafish facility.,transgenic line:Tgfabp10a CreERT2;fabp10a flox beta catenin|age:11 mpf at larval stg:10micromolar 4 hydroxytamoxifen from 3 dpf to 6 dpf,GSM4087862,GSM4087862: 605; Danio rerio; RNA Seq,GSM4087862,,1,Zebrafish were euthanized by rapid chilling and their livers were dissected. Half of each liver was submitted for histologic evaluation to confirm the diagnosis HCC or no HCC. The other half of each dissected liver was flash frozen on dry ice and stored in 80C prior to RNA extraction. RNA was isolated using TriReagent Cat# R2050 1 50 Zymo Research and Direct Zol RNA Mini prep kit Cat# R2050 Zymo Research including DNase treatment following manufacturer's protocols. Samples were eluted in RNase /DNase free water. Sample quality control library preparation sequencing and alignments were performed by the Huntsman Cancer Institute HCI High Throughput Genomics and Bioinformatic Analysis Shared Resource. Paired read 150 base pair sequencing was performed using Illumina's NovaSeq instrument. Three or four pooled libraries were run on each lane of the flow cell. Illumina TruSeq Stranded mRNA Library Prep RIN 8 10,GEO Accession:GSM4087862,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP222804,,,16048X2_190607_A00421_0071_BHL22MDSXX_S2_L004_R1_001.fastq.gz 16048X2_190607_A00421_0071_BHL22MDSXX_S2_L004_R2_001.fastq.gz,fastq fastq,9642566456.0,31929028.0,GSM4087862 r1,0:151 1:151,A:2562227409;C:2265732155;G:2354688485;T:2459736870;N:181537,151,151,,,2562227409,2265732155,2354688485,2459736870,181537,SRX6879042,SRS5414790,SRA965568,GEO,"Pathology, University of California, San Francisco",2,0.9525,0.95448,0.0459,0.04564,0.78173,0.78317,0.54755,0.54727,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,United States,2019-09-20,Multi-stage,Multi-stage,Liver,Liver and Biliary System 55218,SRR10153626,SRX6879041,SRS5414789,SRP222804,PRJNA573071,Bulk transcriptomic analysis of two models of zebrafish beta catenin driven HCC,GSE137787,Transcriptome Analysis,Up to 41% of hepatocellular carcinomas HCCs result from activating mutations in the CTNNB1 gene encoding ß catenin. ß catenin has dual cellular functions as a component of the Wnt signaling pathway and adherens junctions. HCC associated CTNNB1 mutations stabilize the ß catenin protein leading to nuclear and/or cytoplasmic localization of ß catenin and downstream activation of Wnt target genes. In patient HCC samples ß catenin nuclear and cytoplasmic localization are typically patchy even among HCC with highly active CTNNB1 mutations. The functional and clinical relevance of this heterogeneity in ß catenin activation are not well understood. To define mechanisms of ß catenin driven HCC initiation we generated a Cre lox system that enabled switching on activated ß catenin in 1 a small number of hepatocytes in early development; or 2 the majority of hepatocytes in later development or maturity. We discovered that switching on activated ß catenin in a subset of larval hepatocytes was sufficient to drive HCC initiation. To determine the role of Wnt/ß catenin signaling heterogeneity later in hepatocarcinogenesis we performed RNA seq analysis of zebrafish ß catenin driven HCC. Ingenuity Pathway Analysis of differentially expressed genes in the Cre lox HCC model revealed that “Cancer” and “Liver Tumor” categories were significantly altered indicating transcriptional similarities with human HCC and other vertebrate HCC models. At the single cell level 2.9% to 15.2% of hepatocytes from zebrafish ß catenin driven HCC expressed two or more of the Wnt target genes axin2 mtor glula myca and wif1 indicating focal activation of Wnt signaling in established tumors. Thus heterogeneous ß catenin activation drives HCC initiation and persists throughout hepatocarcinogenesis. Overall design: Examination of HCC and nonHCC liver samples from two different models of zebrafish compared to wildtype/nonHCC siblings.,parent bioproject:PRJNA573068,pubmed:31575545,,604,GSM4087861,,tissue:Liver|transgenic line:Tgfabp10a CreERT2;fabp10a flox beta catenin|age:11 mpf at larval stg:10micromolar 4 hydroxytamoxifen from 3 dpf to 6 dpf,604,The Zebrafish GRCz11 FASTA and GTF files were downloaded from Ensembl release 94. The reference database was created using STAR version 2.6.1b with splice junctions optimized for 125 base pair reads.  Reads were trimmed of adapters and aligned to the reference database using STAR in two pass mode to output a BAM file sorted by coordinates.  Mapped reads were assigned to annotated genes in the GTF file using featureCounts version 1.6.3.  Differentially expressed genes were identified using a 5% false discovery rate with DESeq2 version 1.22.2. Genome build: GRCz11 Supplementary files format and content: Raw gene expression quantification with featureCounts v1.6.3 was collated into a tab delimited matrix where each column denotes a sample and each row corresponds to a gene.,Liver,Larvae were treated with either 0.1% ethanol or 10micromolar 4 hydroxytamoxifen from 3 dpf to 6 dpf,Zebrafish were euthanized by rapid chilling and their livers were dissected. Half of each liver was submitted for histologic evaluation to confirm the diagnosis HCC or no HCC. The other half of each dissected liver was flash frozen on dry ice and stored in 80C prior to RNA extraction. RNA was isolated using TriReagent Cat# R2050 1 50 Zymo Research and Direct Zol RNA Mini prep kit Cat# R2050 Zymo Research including DNase treatment following manufacturer’s protocols. Samples were eluted in RNase /DNase free water. Sample quality control library preparation sequencing and alignments were performed by the Huntsman Cancer Institute HCI High Throughput Genomics and Bioinformatic Analysis Shared Resource. Paired read 150 base pair sequencing was performed using Illumina’s NovaSeq instrument. Three or four pooled libraries were run on each lane of the flow cell. Illumina TruSeq Stranded mRNA Library Prep RIN 8 10,Fish were raised following IACUC approved protocols in insititutional zebrafish facility.,transgenic line:Tgfabp10a CreERT2;fabp10a flox beta catenin|age:11 mpf at larval stg:10micromolar 4 hydroxytamoxifen from 3 dpf to 6 dpf,GSM4087861,GSM4087861: 604; Danio rerio; RNA Seq,GSM4087861,,1,Zebrafish were euthanized by rapid chilling and their livers were dissected. Half of each liver was submitted for histologic evaluation to confirm the diagnosis HCC or no HCC. The other half of each dissected liver was flash frozen on dry ice and stored in 80C prior to RNA extraction. RNA was isolated using TriReagent Cat# R2050 1 50 Zymo Research and Direct Zol RNA Mini prep kit Cat# R2050 Zymo Research including DNase treatment following manufacturer's protocols. Samples were eluted in RNase /DNase free water. Sample quality control library preparation sequencing and alignments were performed by the Huntsman Cancer Institute HCI High Throughput Genomics and Bioinformatic Analysis Shared Resource. Paired read 150 base pair sequencing was performed using Illumina's NovaSeq instrument. Three or four pooled libraries were run on each lane of the flow cell. Illumina TruSeq Stranded mRNA Library Prep RIN 8 10,GEO Accession:GSM4087861,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP222804,,,16048X1_190607_A00421_0071_BHL22MDSXX_S3_L004_R1_001.fastq.gz 16048X1_190607_A00421_0071_BHL22MDSXX_S3_L004_R2_001.fastq.gz,fastq fastq,12056607986.0,39922543.0,GSM4087861 r1,0:151 1:151,A:3165745037;C:2870251386;G:2976751806;T:3043633552;N:226205,151,151,,,3165745037,2870251386,2976751806,3043633552,226205,SRX6879041,SRS5414789,SRA965568,GEO,"Pathology, University of California, San Francisco",2,0.95408,0.95527,0.04826,0.04735,0.79366,0.79425,0.52338,0.53718,151,151,B,B,biological fallback assumption,illumina,novaseq_era,unknown,cdna_unspecified,trueseq,bulk,bulk,bulk,,United States,2019-09-20,Multi-stage,Multi-stage,Liver,Liver and Biliary System 59319,SRR11856744,SRX8406857,SRS6719220,SRP264975,PRJNA635397,Transcriptomic profiling of the liver from zebrafish larvae exposed to 175 mM and 350 mM ethanol for xxx hours collected at xxx hpf,GSE151291,Transcriptome Analysis,Exposure to ethanol causes liver injury and activates stress response pathways. Here we compared gene expression in the zebrafish larval liver between larvae that were untreated and those exposed to 175 mM and 350 mM ethanol from xxx 120 hpf. Overall design: We utlized transgenic zebrafish larvae that express a fluorescent protein in the endoplasmic reticulum tgfabp10a: ER tdTomato specifically in hepatocytes as this is used to guide the microdisssection of larval livers. This experiment was conducted by natural spawnings between tg:fabp10:ER tdTomato and WT ABNYU and larvae were exposed from 96 hpf 120 hpf ttwo xxx 175 and 350 mM ethanol. We collected livers from 20 35 larvae for each condition and these were pooled for total RNA extraction using TRIZOL and library preparation using polyA selection as per manufacturer's instructions. Samples were obtained from 5 separate clutches. Samples were sequenced on NextSeq550 Illumina.,,,,5 dpf liver exposed to 350 mM Ethanol clutch 7,GSM4572124,,tissue:Liver|genotype:Tgfabp10a:ER tdTomato|pool:20,5 dpf liver exposed to 350 mM Ethanol clutch 7,Illumina Casava1.8 software used for basecalling. The raw reads were quality assessed using FastQC v0.11.5. The raw reads were then quality trimmed using Trimmomatic trimmomatic adapter.fa:2:30:10 TRAILING:3 LEADING:3 SLIDINGWINDOW:4:15 MINLEN:36 Alignments of trimmed reads were performed using tophat2 v2.1.0 with the parameters “–no novel junctions” and “–G” Accepted bam files were used for counting gene read numbers with HTSeq a 10 s no Test of differential expression of each transposonis was implemented by DESeq2 in Bioconductor Genome build: GRCz10 Supplementary files format and content: .txt Reads count of genes,Liver,At 96 hpf 4 days post the embryos were collected plates were treated with 1% and 2% Ethanol Sigma Aldrich for 24 hour exposure with untreated as control. At 5 dpf 9 am the ethanol was washed out 3X and treated with 500 uM tricaine Ethyl 3 aminobenzoate methanesulfonate Sigma Aldrich USA. Larvae were sorted for red liver positive and these fish were dissected in 3% methyl cellulose.,Larvae were anesthetized with tricane and 20 35 livers were microdissected for each condition and immediately placed into 500 uL of TRIzol Thermo Fisher 15596026.RNA from pooled livers was then extracted per standard TRIzol/Chloroform method and concentrated through isopronanol and resuspended in 20 uL of DNase/RNase free water Thermo Fisher Scientifc. RNA was quantified by Qubit flurometer.,Mating tanks were set up at approximately 4:30 PM the night before and embryos were collected at 10:30 AM the following morning. Following collection embryos were divided into 30 mL plates 60 embryos per plate in egg water at 28C in 10:14 light / dark cycle. Plates were cleaned to remove unfertilized or dead embryos.,genotype:Tgfabp10a:ER tdTomato|pool:20|rna qbit concentrationng/ul:23.8,GSM4572124,GSM4572124: LIV 2ETOH 7; Danio rerio; RNA Seq,GSM4572124,,1,20 embryo livers were pooled per sample and extracted with Trizol as per manufacturer's instructions. RNA seq libraries treated with polyA were prepared by Mehar,GEO Accession:GSM4572124,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 550,,SRP264975,,,LIV_2ETOH_7_S23_read2.fastq.gz LIV_2ETOH_7_S23_read1.fastq.gz,fastq fastq,6357898307.0,22104243.0,GSM4572124 r1,0:143.74 1:143.89,A:1712090294;C:1465925369;G:1489380825;T:1689427333;N:1074486,143,143,,,1712090294,1465925369,1489380825,1689427333,1074486,SRX8406857,SRS6719220,SRA1080364,GEO,"Biology, New York University Abu Dhabi",2,0.69131,0.71792,0.04808,0.0499,0.82477,0.83082,0.47278,0.49809,150,151,B,B,biological fallback assumption,illumina,nextseq,unknown,poly_a,unknown,bulk,bulk,bulk,,United Arab Emirates,2020-05-27,Multi-stage,Multi-stage,Liver,Liver and Biliary System 59320,SRR11856743,SRX8406856,SRS6719219,SRP264975,PRJNA635397,Transcriptomic profiling of the liver from zebrafish larvae exposed to 175 mM and 350 mM ethanol for xxx hours collected at xxx hpf,GSE151291,Transcriptome Analysis,Exposure to ethanol causes liver injury and activates stress response pathways. Here we compared gene expression in the zebrafish larval liver between larvae that were untreated and those exposed to 175 mM and 350 mM ethanol from xxx 120 hpf. Overall design: We utlized transgenic zebrafish larvae that express a fluorescent protein in the endoplasmic reticulum tgfabp10a: ER tdTomato specifically in hepatocytes as this is used to guide the microdisssection of larval livers. This experiment was conducted by natural spawnings between tg:fabp10:ER tdTomato and WT ABNYU and larvae were exposed from 96 hpf 120 hpf ttwo xxx 175 and 350 mM ethanol. We collected livers from 20 35 larvae for each condition and these were pooled for total RNA extraction using TRIZOL and library preparation using polyA selection as per manufacturer's instructions. Samples were obtained from 5 separate clutches. Samples were sequenced on NextSeq550 Illumina.,,,,5 dpf liver exposed to 175mM Ethanol clutch 7,GSM4572123,,tissue:Liver|genotype:Tgfabp10a:ER tdTomato|pool:20,5 dpf liver exposed to 175mM Ethanol clutch 7,Illumina Casava1.8 software used for basecalling. The raw reads were quality assessed using FastQC v0.11.5. The raw reads were then quality trimmed using Trimmomatic trimmomatic adapter.fa:2:30:10 TRAILING:3 LEADING:3 SLIDINGWINDOW:4:15 MINLEN:36 Alignments of trimmed reads were performed using tophat2 v2.1.0 with the parameters “–no novel junctions” and “–G” Accepted bam files were used for counting gene read numbers with HTSeq a 10 s no Test of differential expression of each transposonis was implemented by DESeq2 in Bioconductor Genome build: GRCz10 Supplementary files format and content: .txt Reads count of genes,Liver,At 96 hpf 4 days post the embryos were collected plates were treated with 1% and 2% Ethanol Sigma Aldrich for 24 hour exposure with untreated as control. At 5 dpf 9 am the ethanol was washed out 3X and treated with 500 uM tricaine Ethyl 3 aminobenzoate methanesulfonate Sigma Aldrich USA. Larvae were sorted for red liver positive and these fish were dissected in 3% methyl cellulose.,Larvae were anesthetized with tricane and 20 35 livers were microdissected for each condition and immediately placed into 500 uL of TRIzol Thermo Fisher 15596026.RNA from pooled livers was then extracted per standard TRIzol/Chloroform method and concentrated through isopronanol and resuspended in 20 uL of DNase/RNase free water Thermo Fisher Scientifc. RNA was quantified by Qubit flurometer.,Mating tanks were set up at approximately 4:30 PM the night before and embryos were collected at 10:30 AM the following morning. Following collection embryos were divided into 30 mL plates 60 embryos per plate in egg water at 28C in 10:14 light / dark cycle. Plates were cleaned to remove unfertilized or dead embryos.,genotype:Tgfabp10a:ER tdTomato|pool:20|rna qbit concentrationng/ul:15.7,GSM4572123,GSM4572123: LIV 1ETOH 7; Danio rerio; RNA Seq,GSM4572123,,1,20 embryo livers were pooled per sample and extracted with Trizol as per manufacturer's instructions. RNA seq libraries treated with polyA were prepared by Mehar,GEO Accession:GSM4572123,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 550,,SRP264975,,,LIV_1ETOH_7_S22_read2.fastq.gz LIV_1ETOH_7_S22_read1.fastq.gz,fastq fastq,4305184103.0,15015974.0,GSM4572123 r1,0:143.31 1:143.40,A:1153593916;C:998854522;G:1012290687;T:1139809167;N:635811,143,143,,,1153593916,998854522,1012290687,1139809167,635811,SRX8406856,SRS6719219,SRA1080364,GEO,"Biology, New York University Abu Dhabi",2,0.9174,0.9191,0.06548,0.06503,0.8143,0.82148,0.54687,0.54095,151,151,B,B,biological fallback assumption,illumina,nextseq,unknown,poly_a,unknown,bulk,bulk,bulk,,United Arab Emirates,2020-05-27,Multi-stage,Multi-stage,Liver,Liver and Biliary System 59321,SRR11856756,SRX8406855,SRS6719218,SRP264975,PRJNA635397,Transcriptomic profiling of the liver from zebrafish larvae exposed to 175 mM and 350 mM ethanol for xxx hours collected at xxx hpf,GSE151291,Transcriptome Analysis,Exposure to ethanol causes liver injury and activates stress response pathways. Here we compared gene expression in the zebrafish larval liver between larvae that were untreated and those exposed to 175 mM and 350 mM ethanol from xxx 120 hpf. Overall design: We utlized transgenic zebrafish larvae that express a fluorescent protein in the endoplasmic reticulum tgfabp10a: ER tdTomato specifically in hepatocytes as this is used to guide the microdisssection of larval livers. This experiment was conducted by natural spawnings between tg:fabp10:ER tdTomato and WT ABNYU and larvae were exposed from 96 hpf 120 hpf ttwo xxx 175 and 350 mM ethanol. We collected livers from 20 35 larvae for each condition and these were pooled for total RNA extraction using TRIZOL and library preparation using polyA selection as per manufacturer's instructions. Samples were obtained from 5 separate clutches. Samples were sequenced on NextSeq550 Illumina.,,,,5 dpf Untreated liver clutch 7,GSM4572122,,tissue:Liver|genotype:Tgfabp10a:ER tdTomato|pool:20,5 dpf Untreated liver clutch 7,Illumina Casava1.8 software used for basecalling. The raw reads were quality assessed using FastQC v0.11.5. The raw reads were then quality trimmed using Trimmomatic trimmomatic adapter.fa:2:30:10 TRAILING:3 LEADING:3 SLIDINGWINDOW:4:15 MINLEN:36 Alignments of trimmed reads were performed using tophat2 v2.1.0 with the parameters “–no novel junctions” and “–G” Accepted bam files were used for counting gene read numbers with HTSeq a 10 s no Test of differential expression of each transposonis was implemented by DESeq2 in Bioconductor Genome build: GRCz10 Supplementary files format and content: .txt Reads count of genes,Liver,At 96 hpf 4 days post the embryos were collected plates were treated with 1% and 2% Ethanol Sigma Aldrich for 24 hour exposure with untreated as control. At 5 dpf 9 am the ethanol was washed out 3X and treated with 500 uM tricaine Ethyl 3 aminobenzoate methanesulfonate Sigma Aldrich USA. Larvae were sorted for red liver positive and these fish were dissected in 3% methyl cellulose.,Larvae were anesthetized with tricane and 20 35 livers were microdissected for each condition and immediately placed into 500 uL of TRIzol Thermo Fisher 15596026.RNA from pooled livers was then extracted per standard TRIzol/Chloroform method and concentrated through isopronanol and resuspended in 20 uL of DNase/RNase free water Thermo Fisher Scientifc. RNA was quantified by Qubit flurometer.,Mating tanks were set up at approximately 4:30 PM the night before and embryos were collected at 10:30 AM the following morning. Following collection embryos were divided into 30 mL plates 60 embryos per plate in egg water at 28C in 10:14 light / dark cycle. Plates were cleaned to remove unfertilized or dead embryos.,genotype:Tgfabp10a:ER tdTomato|pool:20|rna qbit concentrationng/ul:14.4,GSM4572122,GSM4572122: LIV UT 7; Danio rerio; RNA Seq,GSM4572122,,1,20 embryo livers were pooled per sample and extracted with Trizol as per manufacturer's instructions. RNA seq libraries treated with polyA were prepared by Mehar,GEO Accession:GSM4572122,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 550,,SRP264975,,,LIV_UT_7_S21_read2.fastq.gz LIV_UT_7_S21_read1.fastq.gz,fastq fastq,4730299683.0,16394224.0,GSM4572122 r1,0:144.23 1:144.30,A:1269896715;C:1097260368;G:1106021062;T:1256450979;N:670559,144,144,,,1269896715,1097260368,1106021062,1256450979,670559,SRX8406855,SRS6719218,SRA1080364,GEO,"Biology, New York University Abu Dhabi",2,0.92607,0.92755,0.06001,0.05999,0.81613,0.82187,0.55335,0.53614,151,151,B,B,biological fallback assumption,illumina,nextseq,unknown,poly_a,unknown,bulk,bulk,bulk,,United Arab Emirates,2020-05-27,Multi-stage,Multi-stage,Liver,Liver and Biliary System 59322,SRR11856755,SRX8406854,SRS6719217,SRP264975,PRJNA635397,Transcriptomic profiling of the liver from zebrafish larvae exposed to 175 mM and 350 mM ethanol for xxx hours collected at xxx hpf,GSE151291,Transcriptome Analysis,Exposure to ethanol causes liver injury and activates stress response pathways. Here we compared gene expression in the zebrafish larval liver between larvae that were untreated and those exposed to 175 mM and 350 mM ethanol from xxx 120 hpf. Overall design: We utlized transgenic zebrafish larvae that express a fluorescent protein in the endoplasmic reticulum tgfabp10a: ER tdTomato specifically in hepatocytes as this is used to guide the microdisssection of larval livers. This experiment was conducted by natural spawnings between tg:fabp10:ER tdTomato and WT ABNYU and larvae were exposed from 96 hpf 120 hpf ttwo xxx 175 and 350 mM ethanol. We collected livers from 20 35 larvae for each condition and these were pooled for total RNA extraction using TRIZOL and library preparation using polyA selection as per manufacturer's instructions. Samples were obtained from 5 separate clutches. Samples were sequenced on NextSeq550 Illumina.,,,,5 dpf liver exposed to 350 mM Ethanol clutch 5,GSM4572121,,tissue:Liver|genotype:Tgfabp10a:ER tdTomato|pool:35,5 dpf liver exposed to 350 mM Ethanol clutch 5,Illumina Casava1.8 software used for basecalling. The raw reads were quality assessed using FastQC v0.11.5. The raw reads were then quality trimmed using Trimmomatic trimmomatic adapter.fa:2:30:10 TRAILING:3 LEADING:3 SLIDINGWINDOW:4:15 MINLEN:36 Alignments of trimmed reads were performed using tophat2 v2.1.0 with the parameters “–no novel junctions” and “–G” Accepted bam files were used for counting gene read numbers with HTSeq a 10 s no Test of differential expression of each transposonis was implemented by DESeq2 in Bioconductor Genome build: GRCz10 Supplementary files format and content: .txt Reads count of genes,Liver,At 96 hpf 4 days post the embryos were collected plates were treated with 1% and 2% Ethanol Sigma Aldrich for 24 hour exposure with untreated as control. At 5 dpf 9 am the ethanol was washed out 3X and treated with 500 uM tricaine Ethyl 3 aminobenzoate methanesulfonate Sigma Aldrich USA. Larvae were sorted for red liver positive and these fish were dissected in 3% methyl cellulose.,Larvae were anesthetized with tricane and 20 35 livers were microdissected for each condition and immediately placed into 500 uL of TRIzol Thermo Fisher 15596026.RNA from pooled livers was then extracted per standard TRIzol/Chloroform method and concentrated through isopronanol and resuspended in 20 uL of DNase/RNase free water Thermo Fisher Scientifc. RNA was quantified by Qubit flurometer.,Mating tanks were set up at approximately 4:30 PM the night before and embryos were collected at 10:30 AM the following morning. Following collection embryos were divided into 30 mL plates 60 embryos per plate in egg water at 28C in 10:14 light / dark cycle. Plates were cleaned to remove unfertilized or dead embryos.,genotype:Tgfabp10a:ER tdTomato|pool:35|rna qbit concentrationng/ul:32.6,GSM4572121,GSM4572121: LIV 2ETOH 5; Danio rerio; RNA Seq,GSM4572121,,1,20 embryo livers were pooled per sample and extracted with Trizol as per manufacturer's instructions. RNA seq libraries treated with polyA were prepared by Mehar,GEO Accession:GSM4572121,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 550,,SRP264975,,,LIV_2ETOH_5_S20_read2.fastq.gz LIV_2ETOH_5_S20_read1.fastq.gz,fastq fastq,5199368627.0,17618880.0,GSM4572121 r1,0:147.54 1:147.56,A:1399517489;C:1172048358;G:1240786042;T:1386342191;N:674547,147,147,,,1399517489,1172048358,1240786042,1386342191,674547,SRX8406854,SRS6719217,SRA1080364,GEO,"Biology, New York University Abu Dhabi",2,0.91793,0.91561,0.14142,0.13588,0.80941,0.81889,0.47417,0.50399,151,150,B,B,biological fallback assumption,illumina,nextseq,unknown,poly_a,unknown,bulk,bulk,bulk,,United Arab Emirates,2020-05-27,Multi-stage,Multi-stage,Liver,Liver and Biliary System 59323,SRR11856754,SRX8406853,SRS6719216,SRP264975,PRJNA635397,Transcriptomic profiling of the liver from zebrafish larvae exposed to 175 mM and 350 mM ethanol for xxx hours collected at xxx hpf,GSE151291,Transcriptome Analysis,Exposure to ethanol causes liver injury and activates stress response pathways. Here we compared gene expression in the zebrafish larval liver between larvae that were untreated and those exposed to 175 mM and 350 mM ethanol from xxx 120 hpf. Overall design: We utlized transgenic zebrafish larvae that express a fluorescent protein in the endoplasmic reticulum tgfabp10a: ER tdTomato specifically in hepatocytes as this is used to guide the microdisssection of larval livers. This experiment was conducted by natural spawnings between tg:fabp10:ER tdTomato and WT ABNYU and larvae were exposed from 96 hpf 120 hpf ttwo xxx 175 and 350 mM ethanol. We collected livers from 20 35 larvae for each condition and these were pooled for total RNA extraction using TRIZOL and library preparation using polyA selection as per manufacturer's instructions. Samples were obtained from 5 separate clutches. Samples were sequenced on NextSeq550 Illumina.,,,,5 dpf liver exposed to 175mM Ethanol clutch 5,GSM4572120,,tissue:Liver|genotype:Tgfabp10a:ER tdTomato|pool:35,5 dpf liver exposed to 175mM Ethanol clutch 5,Illumina Casava1.8 software used for basecalling. The raw reads were quality assessed using FastQC v0.11.5. The raw reads were then quality trimmed using Trimmomatic trimmomatic adapter.fa:2:30:10 TRAILING:3 LEADING:3 SLIDINGWINDOW:4:15 MINLEN:36 Alignments of trimmed reads were performed using tophat2 v2.1.0 with the parameters “–no novel junctions” and “–G” Accepted bam files were used for counting gene read numbers with HTSeq a 10 s no Test of differential expression of each transposonis was implemented by DESeq2 in Bioconductor Genome build: GRCz10 Supplementary files format and content: .txt Reads count of genes,Liver,At 96 hpf 4 days post the embryos were collected plates were treated with 1% and 2% Ethanol Sigma Aldrich for 24 hour exposure with untreated as control. At 5 dpf 9 am the ethanol was washed out 3X and treated with 500 uM tricaine Ethyl 3 aminobenzoate methanesulfonate Sigma Aldrich USA. Larvae were sorted for red liver positive and these fish were dissected in 3% methyl cellulose.,Larvae were anesthetized with tricane and 20 35 livers were microdissected for each condition and immediately placed into 500 uL of TRIzol Thermo Fisher 15596026.RNA from pooled livers was then extracted per standard TRIzol/Chloroform method and concentrated through isopronanol and resuspended in 20 uL of DNase/RNase free water Thermo Fisher Scientifc. RNA was quantified by Qubit flurometer.,Mating tanks were set up at approximately 4:30 PM the night before and embryos were collected at 10:30 AM the following morning. Following collection embryos were divided into 30 mL plates 60 embryos per plate in egg water at 28C in 10:14 light / dark cycle. Plates were cleaned to remove unfertilized or dead embryos.,genotype:Tgfabp10a:ER tdTomato|pool:35|rna qbit concentrationng/ul:34.8,GSM4572120,GSM4572120: LIV 1ETOH 5; Danio rerio; RNA Seq,GSM4572120,,1,20 embryo livers were pooled per sample and extracted with Trizol as per manufacturer's instructions. RNA seq libraries treated with polyA were prepared by Mehar,GEO Accession:GSM4572120,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 550,,SRP264975,,,LIV_1ETOH_5_S19_read2.fastq.gz LIV_1ETOH_5_S19_read1.fastq.gz,fastq fastq,4664825517.0,15773614.0,GSM4572120 r1,0:147.85 1:147.89,A:1254524905;C:1075035904;G:1088910190;T:1245664535;N:689983,147,147,,,1254524905,1075035904,1088910190,1245664535,689983,SRX8406853,SRS6719216,SRA1080364,GEO,"Biology, New York University Abu Dhabi",2,0.94346,0.94595,0.05958,0.05908,0.82183,0.83053,0.53444,0.52504,151,151,B,B,biological fallback assumption,illumina,nextseq,unknown,poly_a,unknown,bulk,bulk,bulk,,United Arab Emirates,2020-05-27,Multi-stage,Multi-stage,Liver,Liver and Biliary System 59324,SRR11856753,SRX8406852,SRS6719215,SRP264975,PRJNA635397,Transcriptomic profiling of the liver from zebrafish larvae exposed to 175 mM and 350 mM ethanol for xxx hours collected at xxx hpf,GSE151291,Transcriptome Analysis,Exposure to ethanol causes liver injury and activates stress response pathways. Here we compared gene expression in the zebrafish larval liver between larvae that were untreated and those exposed to 175 mM and 350 mM ethanol from xxx 120 hpf. Overall design: We utlized transgenic zebrafish larvae that express a fluorescent protein in the endoplasmic reticulum tgfabp10a: ER tdTomato specifically in hepatocytes as this is used to guide the microdisssection of larval livers. This experiment was conducted by natural spawnings between tg:fabp10:ER tdTomato and WT ABNYU and larvae were exposed from 96 hpf 120 hpf ttwo xxx 175 and 350 mM ethanol. We collected livers from 20 35 larvae for each condition and these were pooled for total RNA extraction using TRIZOL and library preparation using polyA selection as per manufacturer's instructions. Samples were obtained from 5 separate clutches. Samples were sequenced on NextSeq550 Illumina.,,,,5 dpf Untreated liver clutch 5,GSM4572119,,tissue:Liver|genotype:Tgfabp10a:ER tdTomato|pool:35,5 dpf Untreated liver clutch 5,Illumina Casava1.8 software used for basecalling. The raw reads were quality assessed using FastQC v0.11.5. The raw reads were then quality trimmed using Trimmomatic trimmomatic adapter.fa:2:30:10 TRAILING:3 LEADING:3 SLIDINGWINDOW:4:15 MINLEN:36 Alignments of trimmed reads were performed using tophat2 v2.1.0 with the parameters “–no novel junctions” and “–G” Accepted bam files were used for counting gene read numbers with HTSeq a 10 s no Test of differential expression of each transposonis was implemented by DESeq2 in Bioconductor Genome build: GRCz10 Supplementary files format and content: .txt Reads count of genes,Liver,At 96 hpf 4 days post the embryos were collected plates were treated with 1% and 2% Ethanol Sigma Aldrich for 24 hour exposure with untreated as control. At 5 dpf 9 am the ethanol was washed out 3X and treated with 500 uM tricaine Ethyl 3 aminobenzoate methanesulfonate Sigma Aldrich USA. Larvae were sorted for red liver positive and these fish were dissected in 3% methyl cellulose.,Larvae were anesthetized with tricane and 20 35 livers were microdissected for each condition and immediately placed into 500 uL of TRIzol Thermo Fisher 15596026.RNA from pooled livers was then extracted per standard TRIzol/Chloroform method and concentrated through isopronanol and resuspended in 20 uL of DNase/RNase free water Thermo Fisher Scientifc. RNA was quantified by Qubit flurometer.,Mating tanks were set up at approximately 4:30 PM the night before and embryos were collected at 10:30 AM the following morning. Following collection embryos were divided into 30 mL plates 60 embryos per plate in egg water at 28C in 10:14 light / dark cycle. Plates were cleaned to remove unfertilized or dead embryos.,genotype:Tgfabp10a:ER tdTomato|pool:35|rna qbit concentrationng/ul:31.2,GSM4572119,GSM4572119: LIV UT 5; Danio rerio; RNA Seq,GSM4572119,,1,20 embryo livers were pooled per sample and extracted with Trizol as per manufacturer's instructions. RNA seq libraries treated with polyA were prepared by Mehar,GEO Accession:GSM4572119,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 550,,SRP264975,,,LIV_UT_5_S18_read2.fastq.gz LIV_UT_5_S18_read1.fastq.gz,fastq fastq,5809354899.0,19717109.0,GSM4572119 r1,0:147.29 1:147.35,A:1543780226;C:1359647607;G:1375859882;T:1529075527;N:991657,147,147,,,1543780226,1359647607,1375859882,1529075527,991657,SRX8406852,SRS6719215,SRA1080364,GEO,"Biology, New York University Abu Dhabi",2,0.94774,0.95056,0.05145,0.05139,0.82079,0.82735,0.52842,0.53743,151,151,B,B,biological fallback assumption,illumina,nextseq,unknown,poly_a,unknown,bulk,bulk,bulk,,United Arab Emirates,2020-05-27,Multi-stage,Multi-stage,Liver,Liver and Biliary System