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 33202,SRR29848463,SRX25346228,SRS22013442,SRP520276,PRJNA1136505,Assessing mechanisms driving phenol isopropylated phosphate IPP induced larval photomotor response deficits in zebrafish,GSE272355,Transcriptome Analysis,Isopropylated phenyl phosphates IPP are an additive organophosphate flame retardant OPFR which has been extensively used in furniture electronics automobiles plastics and children's products to slow down the spread of fire. The processing and distribution of IPP containing products have been prohibited but its continuous leaching from end use products has retained the concern of its toxicity. The present study was designed to evaluate IPP induced developmental toxicity using zebrafish embryos. We first performed range finding experiments with embryonic zebrafish exposed to 0 200 ?M IPP from 6 hpf to 120 hpf and found significant morphological impacts like pericardial edema yolk sac edema and spinal curvature at higher concentrations. Following this relying on secondary analyses of our whole embryo mRNA seq data we quantified neurotransmitters and found significant increase in the levels of dopamine and its metabolite 3 methoxytyramine. We then conducted in vitro retinoic acid receptor RAR signaling assay and noticed significant inhibition of RARa but not RAR? and RAR?. For behavioral readouts we performed larval photomotor response LPR assay at sublethal concentrations and observed hypoactive locomotory behavior in exposed larvae. Whole mount immunohistochemistry for 5 methylcytosine and global DNA methylation assay showed significant IPP induced hypermethylation in situ. Finally based on whole embryo RNA seq data we hypothesized that IPP affects the development of brain and eyes. Firstly we performed global DNA methylation in brain and eyes but did not find significant effects. Then we conducted mRNA sequencing on dissected brains and eyes and found 2 and 135 differentially expressed genes respectively. Gene ontology revealed that IPP affect voltage gated ion channel activity synaptic transmission and neurotransmitter signaling. Collectively our data shows that IPP induces morphological abnormalities and disrupts larval photo motor response potentially through RA inhibition and methylomic regulation. Finally we observed that IPP affects gene expression within the developing eye establishing synaptic transmission vision and muscle contraction as a potential causative factor for LPR responses. Overall design: Zebrafish embryos were exposed to 0 or 0.2 uM IPP from 6 hpf to 120 hpf eyes and brains were dissected out RNA extraction and RNA sequencing using a Novogene Library preparation Pipeline and sequencing on an Novaseq 600 Illumina San Diego CA 2x150 bp 40m reads/sample.,,pubmed:39742644,,IPP 120hpf eye replicate4,GSM8399280,,source name:Dissected eye|tissue:Dissected eye|genotype:wildtype|treatment:IPP|time:120hpf|geo loc name:missing|collection date:missing,IPP 120hpf eye replicate4,Raw data raw reads in fastq format was firstly processed throughin house perl scripts. In this step clean data clean reads was obtainedbyremoving reads containing adapter reads containing ploy N and lowqualityreads from raw data. A Reference genome and gene model annotation files were downloadedfrom genome website directly. Index of the reference genome was built usingHisat2 v2.0.5 and paired end clean reads were aligned to the referencegenome using Hisat2 v2.0.5 FeatureCounts v1.5.0 p3 was used to count the reads numbers mappedto each gene. Then FPKM of each gene was calculated based on the lengthof the gene and reads count mapped to this gene. Differential expression analysisof two conditions/groups two biological replicates per condition was performed using the DESeq2R package 1.20.0. Assembly: GRZ11 Supplementary files format and content: Tab delimited txt file contains FPKM values for each sample,Dissected eye,Embryos were exposed to 0 or 0.2 uM IPP in 0.02% DMSO from 6 hpf to 120 hpf,Eyes and brains were dissected from whole embryos homogenized in RNA zol and RNA extraction was conducted using a Directzol RNA Miniprep Plus kit Zymo. RNA quality and quantity was validated using an Agilent Tapestation and Nanodrop. post fragmentation the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non directional library. For the non directional library it was ready post end repair A tailing adapter ligation size selection amplification and purification For the directional library it was ready post end repair A tailing adapter ligation size selection USER enzyme digestion amplification and purification,Embryos were spawned and maintained in system water,tissue:Dissected eye|genotype:wildtype|treatment:IPP|time:120hpf,GSM8399280,GSM8399280: IPP 120hpf eye replicate4; Danio rerio; RNA Seq,GSM8399280 r1,GSM8399280,1,Eyes and brains were dissected from whole embryos homogenized in RNA zol and RNA extraction was conducted using a Directzol RNA Miniprep Plus kit Zymo. RNA quality and quantity was validated using an Agilent Tapestation and Nanodrop. post fragmentation the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non directional library. For the non directional library it was ready post end repair A tailing adapter ligation size selection amplification and purification For the directional library it was ready post end repair A tailing adapter ligation size selection USER enzyme digestion amplification and purification,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP520276,,,IPP_eye_4_1.fq.gz IPP_eye_4_2.fq.gz,fastq fastq,7072359000.0,23574530.0,GSM8399280 r1,0:150 1:150,A:1896065137;C:1651795911;G:1640296836;T:1877949054;N:6252062,150,150,,,1896065137,1651795911,1640296836,1877949054,6252062,SRX25346228,SRS22013442,SRA1925887,"Dasgupta lab, Biological Sciences, Clemson University","Dasgupta lab, Biological Sciences, Clemson University",2,0.90538,0.89728,0.10331,0.10225,0.70593,0.70688,0.44853,0.44733,150,150,B,B,biological fallback assumption,illumina,novaseq_era,unknown,random_priming,unknown,bulk,unknown,unknown,,United States,2024-07-16,Multi-stage,Multi-stage,Eye,Sensory System 33203,SRR29848464,SRX25346227,SRS22013441,SRP520276,PRJNA1136505,Assessing mechanisms driving phenol isopropylated phosphate IPP induced larval photomotor response deficits in zebrafish,GSE272355,Transcriptome Analysis,Isopropylated phenyl phosphates IPP are an additive organophosphate flame retardant OPFR which has been extensively used in furniture electronics automobiles plastics and children's products to slow down the spread of fire. The processing and distribution of IPP containing products have been prohibited but its continuous leaching from end use products has retained the concern of its toxicity. The present study was designed to evaluate IPP induced developmental toxicity using zebrafish embryos. We first performed range finding experiments with embryonic zebrafish exposed to 0 200 ?M IPP from 6 hpf to 120 hpf and found significant morphological impacts like pericardial edema yolk sac edema and spinal curvature at higher concentrations. Following this relying on secondary analyses of our whole embryo mRNA seq data we quantified neurotransmitters and found significant increase in the levels of dopamine and its metabolite 3 methoxytyramine. We then conducted in vitro retinoic acid receptor RAR signaling assay and noticed significant inhibition of RARa but not RAR? and RAR?. For behavioral readouts we performed larval photomotor response LPR assay at sublethal concentrations and observed hypoactive locomotory behavior in exposed larvae. Whole mount immunohistochemistry for 5 methylcytosine and global DNA methylation assay showed significant IPP induced hypermethylation in situ. Finally based on whole embryo RNA seq data we hypothesized that IPP affects the development of brain and eyes. Firstly we performed global DNA methylation in brain and eyes but did not find significant effects. Then we conducted mRNA sequencing on dissected brains and eyes and found 2 and 135 differentially expressed genes respectively. Gene ontology revealed that IPP affect voltage gated ion channel activity synaptic transmission and neurotransmitter signaling. Collectively our data shows that IPP induces morphological abnormalities and disrupts larval photo motor response potentially through RA inhibition and methylomic regulation. Finally we observed that IPP affects gene expression within the developing eye establishing synaptic transmission vision and muscle contraction as a potential causative factor for LPR responses. Overall design: Zebrafish embryos were exposed to 0 or 0.2 uM IPP from 6 hpf to 120 hpf eyes and brains were dissected out RNA extraction and RNA sequencing using a Novogene Library preparation Pipeline and sequencing on an Novaseq 600 Illumina San Diego CA 2x150 bp 40m reads/sample.,,pubmed:39742644,,IPP 120hpf eye replicate3,GSM8399279,,source name:Dissected eye|tissue:Dissected eye|genotype:wildtype|treatment:IPP|time:120hpf|geo loc name:missing|collection date:missing,IPP 120hpf eye replicate3,Raw data raw reads in fastq format was firstly processed throughin house perl scripts. In this step clean data clean reads was obtainedbyremoving reads containing adapter reads containing ploy N and lowqualityreads from raw data. A Reference genome and gene model annotation files were downloadedfrom genome website directly. Index of the reference genome was built usingHisat2 v2.0.5 and paired end clean reads were aligned to the referencegenome using Hisat2 v2.0.5 FeatureCounts v1.5.0 p3 was used to count the reads numbers mappedto each gene. Then FPKM of each gene was calculated based on the lengthof the gene and reads count mapped to this gene. Differential expression analysisof two conditions/groups two biological replicates per condition was performed using the DESeq2R package 1.20.0. Assembly: GRZ11 Supplementary files format and content: Tab delimited txt file contains FPKM values for each sample,Dissected eye,Embryos were exposed to 0 or 0.2 uM IPP in 0.02% DMSO from 6 hpf to 120 hpf,Eyes and brains were dissected from whole embryos homogenized in RNA zol and RNA extraction was conducted using a Directzol RNA Miniprep Plus kit Zymo. RNA quality and quantity was validated using an Agilent Tapestation and Nanodrop. post fragmentation the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non directional library. For the non directional library it was ready post end repair A tailing adapter ligation size selection amplification and purification For the directional library it was ready post end repair A tailing adapter ligation size selection USER enzyme digestion amplification and purification,Embryos were spawned and maintained in system water,tissue:Dissected eye|genotype:wildtype|treatment:IPP|time:120hpf,GSM8399279,GSM8399279: IPP 120hpf eye replicate3; Danio rerio; RNA Seq,GSM8399279 r1,GSM8399279,1,Eyes and brains were dissected from whole embryos homogenized in RNA zol and RNA extraction was conducted using a Directzol RNA Miniprep Plus kit Zymo. RNA quality and quantity was validated using an Agilent Tapestation and Nanodrop. post fragmentation the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non directional library. For the non directional library it was ready post end repair A tailing adapter ligation size selection amplification and purification For the directional library it was ready post end repair A tailing adapter ligation size selection USER enzyme digestion amplification and purification,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP520276,,,IPP_eye_3_1.fq.gz IPP_eye_3_2.fq.gz,fastq fastq,6537519300.0,21791731.0,GSM8399279 r1,0:150 1:150,A:1760227857;C:1520738786;G:1507946481;T:1742862368;N:5743808,150,150,,,1760227857,1520738786,1507946481,1742862368,5743808,SRX25346227,SRS22013441,SRA1925887,"Dasgupta lab, Biological Sciences, Clemson University","Dasgupta lab, Biological Sciences, Clemson University",2,0.90945,0.90902,0.10894,0.10846,0.70431,0.7055,0.44535,0.43463,150,150,B,B,biological fallback assumption,illumina,novaseq_era,unknown,random_priming,unknown,bulk,unknown,unknown,,United States,2024-07-16,Multi-stage,Multi-stage,Eye,Sensory System 33204,SRR29848465,SRX25346226,SRS22013440,SRP520276,PRJNA1136505,Assessing mechanisms driving phenol isopropylated phosphate IPP induced larval photomotor response deficits in zebrafish,GSE272355,Transcriptome Analysis,Isopropylated phenyl phosphates IPP are an additive organophosphate flame retardant OPFR which has been extensively used in furniture electronics automobiles plastics and children's products to slow down the spread of fire. The processing and distribution of IPP containing products have been prohibited but its continuous leaching from end use products has retained the concern of its toxicity. The present study was designed to evaluate IPP induced developmental toxicity using zebrafish embryos. We first performed range finding experiments with embryonic zebrafish exposed to 0 200 ?M IPP from 6 hpf to 120 hpf and found significant morphological impacts like pericardial edema yolk sac edema and spinal curvature at higher concentrations. Following this relying on secondary analyses of our whole embryo mRNA seq data we quantified neurotransmitters and found significant increase in the levels of dopamine and its metabolite 3 methoxytyramine. We then conducted in vitro retinoic acid receptor RAR signaling assay and noticed significant inhibition of RARa but not RAR? and RAR?. For behavioral readouts we performed larval photomotor response LPR assay at sublethal concentrations and observed hypoactive locomotory behavior in exposed larvae. Whole mount immunohistochemistry for 5 methylcytosine and global DNA methylation assay showed significant IPP induced hypermethylation in situ. Finally based on whole embryo RNA seq data we hypothesized that IPP affects the development of brain and eyes. Firstly we performed global DNA methylation in brain and eyes but did not find significant effects. Then we conducted mRNA sequencing on dissected brains and eyes and found 2 and 135 differentially expressed genes respectively. Gene ontology revealed that IPP affect voltage gated ion channel activity synaptic transmission and neurotransmitter signaling. Collectively our data shows that IPP induces morphological abnormalities and disrupts larval photo motor response potentially through RA inhibition and methylomic regulation. Finally we observed that IPP affects gene expression within the developing eye establishing synaptic transmission vision and muscle contraction as a potential causative factor for LPR responses. Overall design: Zebrafish embryos were exposed to 0 or 0.2 uM IPP from 6 hpf to 120 hpf eyes and brains were dissected out RNA extraction and RNA sequencing using a Novogene Library preparation Pipeline and sequencing on an Novaseq 600 Illumina San Diego CA 2x150 bp 40m reads/sample.,,pubmed:39742644,,IPP 120hpf eye replicate2,GSM8399278,,source name:Dissected eye|tissue:Dissected eye|genotype:wildtype|treatment:IPP|time:120hpf|geo loc name:missing|collection date:missing,IPP 120hpf eye replicate2,Raw data raw reads in fastq format was firstly processed throughin house perl scripts. In this step clean data clean reads was obtainedbyremoving reads containing adapter reads containing ploy N and lowqualityreads from raw data. A Reference genome and gene model annotation files were downloadedfrom genome website directly. Index of the reference genome was built usingHisat2 v2.0.5 and paired end clean reads were aligned to the referencegenome using Hisat2 v2.0.5 FeatureCounts v1.5.0 p3 was used to count the reads numbers mappedto each gene. Then FPKM of each gene was calculated based on the lengthof the gene and reads count mapped to this gene. Differential expression analysisof two conditions/groups two biological replicates per condition was performed using the DESeq2R package 1.20.0. Assembly: GRZ11 Supplementary files format and content: Tab delimited txt file contains FPKM values for each sample,Dissected eye,Embryos were exposed to 0 or 0.2 uM IPP in 0.02% DMSO from 6 hpf to 120 hpf,Eyes and brains were dissected from whole embryos homogenized in RNA zol and RNA extraction was conducted using a Directzol RNA Miniprep Plus kit Zymo. RNA quality and quantity was validated using an Agilent Tapestation and Nanodrop. post fragmentation the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non directional library. For the non directional library it was ready post end repair A tailing adapter ligation size selection amplification and purification For the directional library it was ready post end repair A tailing adapter ligation size selection USER enzyme digestion amplification and purification,Embryos were spawned and maintained in system water,tissue:Dissected eye|genotype:wildtype|treatment:IPP|time:120hpf,GSM8399278,GSM8399278: IPP 120hpf eye replicate2; Danio rerio; RNA Seq,GSM8399278 r1,GSM8399278,1,Eyes and brains were dissected from whole embryos homogenized in RNA zol and RNA extraction was conducted using a Directzol RNA Miniprep Plus kit Zymo. RNA quality and quantity was validated using an Agilent Tapestation and Nanodrop. post fragmentation the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non directional library. For the non directional library it was ready post end repair A tailing adapter ligation size selection amplification and purification For the directional library it was ready post end repair A tailing adapter ligation size selection USER enzyme digestion amplification and purification,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP520276,,,IPP_eye_2_1.fq.gz IPP_eye_2_2.fq.gz,fastq fastq,7374435900.0,24581453.0,GSM8399278 r1,0:150 1:150,A:1986621253;C:1713862328;G:1699590477;T:1967874898;N:6486944,150,150,,,1986621253,1713862328,1699590477,1967874898,6486944,SRX25346226,SRS22013440,SRA1925887,"Dasgupta lab, Biological Sciences, Clemson University","Dasgupta lab, Biological Sciences, Clemson University",2,0.9091,0.90128,0.11122,0.1097,0.70201,0.70266,0.44395,0.4416,150,150,B,B,biological fallback assumption,illumina,novaseq_era,unknown,random_priming,unknown,bulk,unknown,unknown,,United States,2024-07-16,Multi-stage,Multi-stage,Eye,Sensory System 33205,SRR29848466,SRX25346225,SRS22013439,SRP520276,PRJNA1136505,Assessing mechanisms driving phenol isopropylated phosphate IPP induced larval photomotor response deficits in zebrafish,GSE272355,Transcriptome Analysis,Isopropylated phenyl phosphates IPP are an additive organophosphate flame retardant OPFR which has been extensively used in furniture electronics automobiles plastics and children's products to slow down the spread of fire. The processing and distribution of IPP containing products have been prohibited but its continuous leaching from end use products has retained the concern of its toxicity. The present study was designed to evaluate IPP induced developmental toxicity using zebrafish embryos. We first performed range finding experiments with embryonic zebrafish exposed to 0 200 ?M IPP from 6 hpf to 120 hpf and found significant morphological impacts like pericardial edema yolk sac edema and spinal curvature at higher concentrations. Following this relying on secondary analyses of our whole embryo mRNA seq data we quantified neurotransmitters and found significant increase in the levels of dopamine and its metabolite 3 methoxytyramine. We then conducted in vitro retinoic acid receptor RAR signaling assay and noticed significant inhibition of RARa but not RAR? and RAR?. For behavioral readouts we performed larval photomotor response LPR assay at sublethal concentrations and observed hypoactive locomotory behavior in exposed larvae. Whole mount immunohistochemistry for 5 methylcytosine and global DNA methylation assay showed significant IPP induced hypermethylation in situ. Finally based on whole embryo RNA seq data we hypothesized that IPP affects the development of brain and eyes. Firstly we performed global DNA methylation in brain and eyes but did not find significant effects. Then we conducted mRNA sequencing on dissected brains and eyes and found 2 and 135 differentially expressed genes respectively. Gene ontology revealed that IPP affect voltage gated ion channel activity synaptic transmission and neurotransmitter signaling. Collectively our data shows that IPP induces morphological abnormalities and disrupts larval photo motor response potentially through RA inhibition and methylomic regulation. Finally we observed that IPP affects gene expression within the developing eye establishing synaptic transmission vision and muscle contraction as a potential causative factor for LPR responses. Overall design: Zebrafish embryos were exposed to 0 or 0.2 uM IPP from 6 hpf to 120 hpf eyes and brains were dissected out RNA extraction and RNA sequencing using a Novogene Library preparation Pipeline and sequencing on an Novaseq 600 Illumina San Diego CA 2x150 bp 40m reads/sample.,,pubmed:39742644,,IPP 120hpf eye replicate1,GSM8399277,,source name:Dissected eye|tissue:Dissected eye|genotype:wildtype|treatment:IPP|time:120hpf|geo loc name:missing|collection date:missing,IPP 120hpf eye replicate1,Raw data raw reads in fastq format was firstly processed throughin house perl scripts. In this step clean data clean reads was obtainedbyremoving reads containing adapter reads containing ploy N and lowqualityreads from raw data. A Reference genome and gene model annotation files were downloadedfrom genome website directly. Index of the reference genome was built usingHisat2 v2.0.5 and paired end clean reads were aligned to the referencegenome using Hisat2 v2.0.5 FeatureCounts v1.5.0 p3 was used to count the reads numbers mappedto each gene. Then FPKM of each gene was calculated based on the lengthof the gene and reads count mapped to this gene. Differential expression analysisof two conditions/groups two biological replicates per condition was performed using the DESeq2R package 1.20.0. Assembly: GRZ11 Supplementary files format and content: Tab delimited txt file contains FPKM values for each sample,Dissected eye,Embryos were exposed to 0 or 0.2 uM IPP in 0.02% DMSO from 6 hpf to 120 hpf,Eyes and brains were dissected from whole embryos homogenized in RNA zol and RNA extraction was conducted using a Directzol RNA Miniprep Plus kit Zymo. RNA quality and quantity was validated using an Agilent Tapestation and Nanodrop. post fragmentation the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non directional library. For the non directional library it was ready post end repair A tailing adapter ligation size selection amplification and purification For the directional library it was ready post end repair A tailing adapter ligation size selection USER enzyme digestion amplification and purification,Embryos were spawned and maintained in system water,tissue:Dissected eye|genotype:wildtype|treatment:IPP|time:120hpf,GSM8399277,GSM8399277: IPP 120hpf eye replicate1; Danio rerio; RNA Seq,GSM8399277 r1,GSM8399277,1,Eyes and brains were dissected from whole embryos homogenized in RNA zol and RNA extraction was conducted using a Directzol RNA Miniprep Plus kit Zymo. RNA quality and quantity was validated using an Agilent Tapestation and Nanodrop. post fragmentation the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non directional library. For the non directional library it was ready post end repair A tailing adapter ligation size selection amplification and purification For the directional library it was ready post end repair A tailing adapter ligation size selection USER enzyme digestion amplification and purification,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP520276,,,IPP_eye_1_1.fq.gz IPP_eye_1_2.fq.gz,fastq fastq,6538011600.0,21793372.0,GSM8399277 r1,0:150 1:150,A:1758764189;C:1520242338;G:1510361700;T:1742917780;N:5725593,150,150,,,1758764189,1520242338,1510361700,1742917780,5725593,SRX25346225,SRS22013439,SRA1925887,"Dasgupta lab, Biological Sciences, Clemson University","Dasgupta lab, Biological Sciences, Clemson University",2,0.91402,0.90585,0.11702,0.11558,0.70029,0.70094,0.45385,0.45435,150,150,B,B,biological fallback assumption,illumina,novaseq_era,unknown,random_priming,unknown,bulk,unknown,unknown,,United States,2024-07-16,Multi-stage,Multi-stage,Eye,Sensory System 33206,SRR29848467,SRX25346224,SRS22013438,SRP520276,PRJNA1136505,Assessing mechanisms driving phenol isopropylated phosphate IPP induced larval photomotor response deficits in zebrafish,GSE272355,Transcriptome Analysis,Isopropylated phenyl phosphates IPP are an additive organophosphate flame retardant OPFR which has been extensively used in furniture electronics automobiles plastics and children's products to slow down the spread of fire. The processing and distribution of IPP containing products have been prohibited but its continuous leaching from end use products has retained the concern of its toxicity. The present study was designed to evaluate IPP induced developmental toxicity using zebrafish embryos. We first performed range finding experiments with embryonic zebrafish exposed to 0 200 ?M IPP from 6 hpf to 120 hpf and found significant morphological impacts like pericardial edema yolk sac edema and spinal curvature at higher concentrations. Following this relying on secondary analyses of our whole embryo mRNA seq data we quantified neurotransmitters and found significant increase in the levels of dopamine and its metabolite 3 methoxytyramine. We then conducted in vitro retinoic acid receptor RAR signaling assay and noticed significant inhibition of RARa but not RAR? and RAR?. For behavioral readouts we performed larval photomotor response LPR assay at sublethal concentrations and observed hypoactive locomotory behavior in exposed larvae. Whole mount immunohistochemistry for 5 methylcytosine and global DNA methylation assay showed significant IPP induced hypermethylation in situ. Finally based on whole embryo RNA seq data we hypothesized that IPP affects the development of brain and eyes. Firstly we performed global DNA methylation in brain and eyes but did not find significant effects. Then we conducted mRNA sequencing on dissected brains and eyes and found 2 and 135 differentially expressed genes respectively. Gene ontology revealed that IPP affect voltage gated ion channel activity synaptic transmission and neurotransmitter signaling. Collectively our data shows that IPP induces morphological abnormalities and disrupts larval photo motor response potentially through RA inhibition and methylomic regulation. Finally we observed that IPP affects gene expression within the developing eye establishing synaptic transmission vision and muscle contraction as a potential causative factor for LPR responses. Overall design: Zebrafish embryos were exposed to 0 or 0.2 uM IPP from 6 hpf to 120 hpf eyes and brains were dissected out RNA extraction and RNA sequencing using a Novogene Library preparation Pipeline and sequencing on an Novaseq 600 Illumina San Diego CA 2x150 bp 40m reads/sample.,,pubmed:39742644,,DMSO 120hpf eye replicate4,GSM8399276,,source name:Dissected eye|tissue:Dissected eye|genotype:wildtype|treatment:DMSO|time:120hpf|geo loc name:missing|collection date:missing,DMSO 120hpf eye replicate4,Raw data raw reads in fastq format was firstly processed throughin house perl scripts. In this step clean data clean reads was obtainedbyremoving reads containing adapter reads containing ploy N and lowqualityreads from raw data. A Reference genome and gene model annotation files were downloadedfrom genome website directly. Index of the reference genome was built usingHisat2 v2.0.5 and paired end clean reads were aligned to the referencegenome using Hisat2 v2.0.5 FeatureCounts v1.5.0 p3 was used to count the reads numbers mappedto each gene. Then FPKM of each gene was calculated based on the lengthof the gene and reads count mapped to this gene. Differential expression analysisof two conditions/groups two biological replicates per condition was performed using the DESeq2R package 1.20.0. Assembly: GRZ11 Supplementary files format and content: Tab delimited txt file contains FPKM values for each sample,Dissected eye,Embryos were exposed to 0 or 0.2 uM IPP in 0.02% DMSO from 6 hpf to 120 hpf,Eyes and brains were dissected from whole embryos homogenized in RNA zol and RNA extraction was conducted using a Directzol RNA Miniprep Plus kit Zymo. RNA quality and quantity was validated using an Agilent Tapestation and Nanodrop. post fragmentation the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non directional library. For the non directional library it was ready post end repair A tailing adapter ligation size selection amplification and purification For the directional library it was ready post end repair A tailing adapter ligation size selection USER enzyme digestion amplification and purification,Embryos were spawned and maintained in system water,tissue:Dissected eye|genotype:wildtype|treatment:DMSO|time:120hpf,GSM8399276,GSM8399276: DMSO 120hpf eye replicate4; Danio rerio; RNA Seq,GSM8399276 r1,GSM8399276,1,Eyes and brains were dissected from whole embryos homogenized in RNA zol and RNA extraction was conducted using a Directzol RNA Miniprep Plus kit Zymo. RNA quality and quantity was validated using an Agilent Tapestation and Nanodrop. post fragmentation the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non directional library. For the non directional library it was ready post end repair A tailing adapter ligation size selection amplification and purification For the directional library it was ready post end repair A tailing adapter ligation size selection USER enzyme digestion amplification and purification,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP520276,,,Ctrl_eye_4_1.fq.gz Ctrl_eye_4_2.fq.gz,fastq fastq,7198057200.0,23993524.0,GSM8399276 r1,0:150 1:150,A:1929066670;C:1680279530;G:1672121671;T:1910263876;N:6325453,150,150,,,1929066670,1680279530,1672121671,1910263876,6325453,SRX25346224,SRS22013438,SRA1925887,"Dasgupta lab, Biological Sciences, Clemson University","Dasgupta lab, Biological Sciences, Clemson University",2,0.91947,0.90384,0.11309,0.11094,0.69576,0.69779,0.45803,0.45394,150,150,B,B,biological fallback assumption,illumina,novaseq_era,unknown,random_priming,unknown,bulk,unknown,unknown,,United States,2024-07-16,Multi-stage,Multi-stage,Eye,Sensory System 33207,SRR29848468,SRX25346223,SRS22013437,SRP520276,PRJNA1136505,Assessing mechanisms driving phenol isopropylated phosphate IPP induced larval photomotor response deficits in zebrafish,GSE272355,Transcriptome Analysis,Isopropylated phenyl phosphates IPP are an additive organophosphate flame retardant OPFR which has been extensively used in furniture electronics automobiles plastics and children's products to slow down the spread of fire. The processing and distribution of IPP containing products have been prohibited but its continuous leaching from end use products has retained the concern of its toxicity. The present study was designed to evaluate IPP induced developmental toxicity using zebrafish embryos. We first performed range finding experiments with embryonic zebrafish exposed to 0 200 ?M IPP from 6 hpf to 120 hpf and found significant morphological impacts like pericardial edema yolk sac edema and spinal curvature at higher concentrations. Following this relying on secondary analyses of our whole embryo mRNA seq data we quantified neurotransmitters and found significant increase in the levels of dopamine and its metabolite 3 methoxytyramine. We then conducted in vitro retinoic acid receptor RAR signaling assay and noticed significant inhibition of RARa but not RAR? and RAR?. For behavioral readouts we performed larval photomotor response LPR assay at sublethal concentrations and observed hypoactive locomotory behavior in exposed larvae. Whole mount immunohistochemistry for 5 methylcytosine and global DNA methylation assay showed significant IPP induced hypermethylation in situ. Finally based on whole embryo RNA seq data we hypothesized that IPP affects the development of brain and eyes. Firstly we performed global DNA methylation in brain and eyes but did not find significant effects. Then we conducted mRNA sequencing on dissected brains and eyes and found 2 and 135 differentially expressed genes respectively. Gene ontology revealed that IPP affect voltage gated ion channel activity synaptic transmission and neurotransmitter signaling. Collectively our data shows that IPP induces morphological abnormalities and disrupts larval photo motor response potentially through RA inhibition and methylomic regulation. Finally we observed that IPP affects gene expression within the developing eye establishing synaptic transmission vision and muscle contraction as a potential causative factor for LPR responses. Overall design: Zebrafish embryos were exposed to 0 or 0.2 uM IPP from 6 hpf to 120 hpf eyes and brains were dissected out RNA extraction and RNA sequencing using a Novogene Library preparation Pipeline and sequencing on an Novaseq 600 Illumina San Diego CA 2x150 bp 40m reads/sample.,,pubmed:39742644,,DMSO 120hpf eye replicate3,GSM8399275,,source name:Dissected eye|tissue:Dissected eye|genotype:wildtype|treatment:DMSO|time:120hpf|geo loc name:missing|collection date:missing,DMSO 120hpf eye replicate3,Raw data raw reads in fastq format was firstly processed throughin house perl scripts. In this step clean data clean reads was obtainedbyremoving reads containing adapter reads containing ploy N and lowqualityreads from raw data. A Reference genome and gene model annotation files were downloadedfrom genome website directly. Index of the reference genome was built usingHisat2 v2.0.5 and paired end clean reads were aligned to the referencegenome using Hisat2 v2.0.5 FeatureCounts v1.5.0 p3 was used to count the reads numbers mappedto each gene. Then FPKM of each gene was calculated based on the lengthof the gene and reads count mapped to this gene. Differential expression analysisof two conditions/groups two biological replicates per condition was performed using the DESeq2R package 1.20.0. Assembly: GRZ11 Supplementary files format and content: Tab delimited txt file contains FPKM values for each sample,Dissected eye,Embryos were exposed to 0 or 0.2 uM IPP in 0.02% DMSO from 6 hpf to 120 hpf,Eyes and brains were dissected from whole embryos homogenized in RNA zol and RNA extraction was conducted using a Directzol RNA Miniprep Plus kit Zymo. RNA quality and quantity was validated using an Agilent Tapestation and Nanodrop. post fragmentation the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non directional library. For the non directional library it was ready post end repair A tailing adapter ligation size selection amplification and purification For the directional library it was ready post end repair A tailing adapter ligation size selection USER enzyme digestion amplification and purification,Embryos were spawned and maintained in system water,tissue:Dissected eye|genotype:wildtype|treatment:DMSO|time:120hpf,GSM8399275,GSM8399275: DMSO 120hpf eye replicate3; Danio rerio; RNA Seq,GSM8399275 r1,GSM8399275,1,Eyes and brains were dissected from whole embryos homogenized in RNA zol and RNA extraction was conducted using a Directzol RNA Miniprep Plus kit Zymo. RNA quality and quantity was validated using an Agilent Tapestation and Nanodrop. post fragmentation the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non directional library. For the non directional library it was ready post end repair A tailing adapter ligation size selection amplification and purification For the directional library it was ready post end repair A tailing adapter ligation size selection USER enzyme digestion amplification and purification,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP520276,,,Ctrl_eye_3_1.fq.gz Ctrl_eye_3_2.fq.gz,fastq fastq,6349681200.0,21165604.0,GSM8399275 r1,0:150 1:150,A:1713020668;C:1474226584;G:1462505802;T:1694350186;N:5577960,150,150,,,1713020668,1474226584,1462505802,1694350186,5577960,SRX25346223,SRS22013437,SRA1925887,"Dasgupta lab, Biological Sciences, Clemson University","Dasgupta lab, Biological Sciences, Clemson University",2,0.89548,0.89606,0.09628,0.09664,0.71102,0.71161,0.44199,0.4354,150,150,B,B,biological fallback assumption,illumina,novaseq_era,unknown,random_priming,unknown,bulk,unknown,unknown,,United States,2024-07-16,Multi-stage,Multi-stage,Eye,Sensory System 33208,SRR29848469,SRX25346222,SRS22013436,SRP520276,PRJNA1136505,Assessing mechanisms driving phenol isopropylated phosphate IPP induced larval photomotor response deficits in zebrafish,GSE272355,Transcriptome Analysis,Isopropylated phenyl phosphates IPP are an additive organophosphate flame retardant OPFR which has been extensively used in furniture electronics automobiles plastics and children's products to slow down the spread of fire. The processing and distribution of IPP containing products have been prohibited but its continuous leaching from end use products has retained the concern of its toxicity. The present study was designed to evaluate IPP induced developmental toxicity using zebrafish embryos. We first performed range finding experiments with embryonic zebrafish exposed to 0 200 ?M IPP from 6 hpf to 120 hpf and found significant morphological impacts like pericardial edema yolk sac edema and spinal curvature at higher concentrations. Following this relying on secondary analyses of our whole embryo mRNA seq data we quantified neurotransmitters and found significant increase in the levels of dopamine and its metabolite 3 methoxytyramine. We then conducted in vitro retinoic acid receptor RAR signaling assay and noticed significant inhibition of RARa but not RAR? and RAR?. For behavioral readouts we performed larval photomotor response LPR assay at sublethal concentrations and observed hypoactive locomotory behavior in exposed larvae. Whole mount immunohistochemistry for 5 methylcytosine and global DNA methylation assay showed significant IPP induced hypermethylation in situ. Finally based on whole embryo RNA seq data we hypothesized that IPP affects the development of brain and eyes. Firstly we performed global DNA methylation in brain and eyes but did not find significant effects. Then we conducted mRNA sequencing on dissected brains and eyes and found 2 and 135 differentially expressed genes respectively. Gene ontology revealed that IPP affect voltage gated ion channel activity synaptic transmission and neurotransmitter signaling. Collectively our data shows that IPP induces morphological abnormalities and disrupts larval photo motor response potentially through RA inhibition and methylomic regulation. Finally we observed that IPP affects gene expression within the developing eye establishing synaptic transmission vision and muscle contraction as a potential causative factor for LPR responses. Overall design: Zebrafish embryos were exposed to 0 or 0.2 uM IPP from 6 hpf to 120 hpf eyes and brains were dissected out RNA extraction and RNA sequencing using a Novogene Library preparation Pipeline and sequencing on an Novaseq 600 Illumina San Diego CA 2x150 bp 40m reads/sample.,,pubmed:39742644,,DMSO 120hpf eye replicate2,GSM8399274,,source name:Dissected eye|tissue:Dissected eye|genotype:wildtype|treatment:DMSO|time:120hpf|geo loc name:missing|collection date:missing,DMSO 120hpf eye replicate2,Raw data raw reads in fastq format was firstly processed throughin house perl scripts. In this step clean data clean reads was obtainedbyremoving reads containing adapter reads containing ploy N and lowqualityreads from raw data. A Reference genome and gene model annotation files were downloadedfrom genome website directly. Index of the reference genome was built usingHisat2 v2.0.5 and paired end clean reads were aligned to the referencegenome using Hisat2 v2.0.5 FeatureCounts v1.5.0 p3 was used to count the reads numbers mappedto each gene. Then FPKM of each gene was calculated based on the lengthof the gene and reads count mapped to this gene. Differential expression analysisof two conditions/groups two biological replicates per condition was performed using the DESeq2R package 1.20.0. Assembly: GRZ11 Supplementary files format and content: Tab delimited txt file contains FPKM values for each sample,Dissected eye,Embryos were exposed to 0 or 0.2 uM IPP in 0.02% DMSO from 6 hpf to 120 hpf,Eyes and brains were dissected from whole embryos homogenized in RNA zol and RNA extraction was conducted using a Directzol RNA Miniprep Plus kit Zymo. RNA quality and quantity was validated using an Agilent Tapestation and Nanodrop. post fragmentation the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non directional library. For the non directional library it was ready post end repair A tailing adapter ligation size selection amplification and purification For the directional library it was ready post end repair A tailing adapter ligation size selection USER enzyme digestion amplification and purification,Embryos were spawned and maintained in system water,tissue:Dissected eye|genotype:wildtype|treatment:DMSO|time:120hpf,GSM8399274,GSM8399274: DMSO 120hpf eye replicate2; Danio rerio; RNA Seq,GSM8399274 r1,GSM8399274,1,Eyes and brains were dissected from whole embryos homogenized in RNA zol and RNA extraction was conducted using a Directzol RNA Miniprep Plus kit Zymo. RNA quality and quantity was validated using an Agilent Tapestation and Nanodrop. post fragmentation the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non directional library. For the non directional library it was ready post end repair A tailing adapter ligation size selection amplification and purification For the directional library it was ready post end repair A tailing adapter ligation size selection USER enzyme digestion amplification and purification,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP520276,,,Ctrl_eye_2_1.fq.gz Ctrl_eye_2_2.fq.gz,fastq fastq,6861299400.0,22870998.0,GSM8399274 r1,0:150 1:150,A:1839006136;C:1604484415;G:1591747628;T:1820307442;N:5753779,150,150,,,1839006136,1604484415,1591747628,1820307442,5753779,SRX25346222,SRS22013436,SRA1925887,"Dasgupta lab, Biological Sciences, Clemson University","Dasgupta lab, Biological Sciences, Clemson University",2,0.84164,0.83344,0.09914,0.09758,0.72034,0.72088,0.44273,0.4389,150,150,B,B,biological fallback assumption,illumina,novaseq_era,unknown,random_priming,unknown,bulk,unknown,unknown,,United States,2024-07-16,Multi-stage,Multi-stage,Eye,Sensory System 33209,SRR29848470,SRX25346221,SRS22013435,SRP520276,PRJNA1136505,Assessing mechanisms driving phenol isopropylated phosphate IPP induced larval photomotor response deficits in zebrafish,GSE272355,Transcriptome Analysis,Isopropylated phenyl phosphates IPP are an additive organophosphate flame retardant OPFR which has been extensively used in furniture electronics automobiles plastics and children's products to slow down the spread of fire. The processing and distribution of IPP containing products have been prohibited but its continuous leaching from end use products has retained the concern of its toxicity. The present study was designed to evaluate IPP induced developmental toxicity using zebrafish embryos. We first performed range finding experiments with embryonic zebrafish exposed to 0 200 ?M IPP from 6 hpf to 120 hpf and found significant morphological impacts like pericardial edema yolk sac edema and spinal curvature at higher concentrations. Following this relying on secondary analyses of our whole embryo mRNA seq data we quantified neurotransmitters and found significant increase in the levels of dopamine and its metabolite 3 methoxytyramine. We then conducted in vitro retinoic acid receptor RAR signaling assay and noticed significant inhibition of RARa but not RAR? and RAR?. For behavioral readouts we performed larval photomotor response LPR assay at sublethal concentrations and observed hypoactive locomotory behavior in exposed larvae. Whole mount immunohistochemistry for 5 methylcytosine and global DNA methylation assay showed significant IPP induced hypermethylation in situ. Finally based on whole embryo RNA seq data we hypothesized that IPP affects the development of brain and eyes. Firstly we performed global DNA methylation in brain and eyes but did not find significant effects. Then we conducted mRNA sequencing on dissected brains and eyes and found 2 and 135 differentially expressed genes respectively. Gene ontology revealed that IPP affect voltage gated ion channel activity synaptic transmission and neurotransmitter signaling. Collectively our data shows that IPP induces morphological abnormalities and disrupts larval photo motor response potentially through RA inhibition and methylomic regulation. Finally we observed that IPP affects gene expression within the developing eye establishing synaptic transmission vision and muscle contraction as a potential causative factor for LPR responses. Overall design: Zebrafish embryos were exposed to 0 or 0.2 uM IPP from 6 hpf to 120 hpf eyes and brains were dissected out RNA extraction and RNA sequencing using a Novogene Library preparation Pipeline and sequencing on an Novaseq 600 Illumina San Diego CA 2x150 bp 40m reads/sample.,,pubmed:39742644,,DMSO 120hpf eye replicate1,GSM8399273,,source name:Dissected eye|tissue:Dissected eye|genotype:wildtype|treatment:DMSO|time:120hpf|geo loc name:missing|collection date:missing,DMSO 120hpf eye replicate1,Raw data raw reads in fastq format was firstly processed throughin house perl scripts. In this step clean data clean reads was obtainedbyremoving reads containing adapter reads containing ploy N and lowqualityreads from raw data. A Reference genome and gene model annotation files were downloadedfrom genome website directly. Index of the reference genome was built usingHisat2 v2.0.5 and paired end clean reads were aligned to the referencegenome using Hisat2 v2.0.5 FeatureCounts v1.5.0 p3 was used to count the reads numbers mappedto each gene. Then FPKM of each gene was calculated based on the lengthof the gene and reads count mapped to this gene. Differential expression analysisof two conditions/groups two biological replicates per condition was performed using the DESeq2R package 1.20.0. Assembly: GRZ11 Supplementary files format and content: Tab delimited txt file contains FPKM values for each sample,Dissected eye,Embryos were exposed to 0 or 0.2 uM IPP in 0.02% DMSO from 6 hpf to 120 hpf,Eyes and brains were dissected from whole embryos homogenized in RNA zol and RNA extraction was conducted using a Directzol RNA Miniprep Plus kit Zymo. RNA quality and quantity was validated using an Agilent Tapestation and Nanodrop. post fragmentation the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non directional library. For the non directional library it was ready post end repair A tailing adapter ligation size selection amplification and purification For the directional library it was ready post end repair A tailing adapter ligation size selection USER enzyme digestion amplification and purification,Embryos were spawned and maintained in system water,tissue:Dissected eye|genotype:wildtype|treatment:DMSO|time:120hpf,GSM8399273,GSM8399273: DMSO 120hpf eye replicate1; Danio rerio; RNA Seq,GSM8399273 r1,GSM8399273,1,Eyes and brains were dissected from whole embryos homogenized in RNA zol and RNA extraction was conducted using a Directzol RNA Miniprep Plus kit Zymo. RNA quality and quantity was validated using an Agilent Tapestation and Nanodrop. post fragmentation the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non directional library. For the non directional library it was ready post end repair A tailing adapter ligation size selection amplification and purification For the directional library it was ready post end repair A tailing adapter ligation size selection USER enzyme digestion amplification and purification,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP520276,,,Ctrl_eye_1_1.fq.gz Ctrl_eye_1_2.fq.gz,fastq fastq,6544491000.0,21814970.0,GSM8399273 r1,0:150 1:150,A:1774622867;C:1510819173;G:1498655117;T:1754689449;N:5704394,150,150,,,1774622867,1510819173,1498655117,1754689449,5704394,SRX25346221,SRS22013435,SRA1925887,"Dasgupta lab, Biological Sciences, Clemson University","Dasgupta lab, Biological Sciences, Clemson University",2,0.86147,0.86095,0.11217,0.11159,0.71319,0.71352,0.44434,0.43886,150,150,B,B,biological fallback assumption,illumina,novaseq_era,unknown,random_priming,unknown,bulk,unknown,unknown,,United States,2024-07-16,Multi-stage,Multi-stage,Eye,Sensory System 34264,SRR31620789,SRX26985240,SRS23451898,SRP549631,PRJNA1194578,Tet proteins regulate differentiation potential across retinal cell types,GSE283588,Transcriptome Analysis,Tet enzymes are epigenetic modifiers that impact gene expression via 5mC to 5hmC oxidation; 5hmC generation is known to induce chromatin remodeling and promote transcriptional accessibility and therefore gene expression. Previous work demonstrated the requirement for Tet and 5hmC in early retinogenesis–revealing that Tet nulltet2 / ;tet3 / zebrafish exhibit extensive retinal defects. Here we leveraged scRNAseq technologies to better understand cell type specific deficits and molecular signatures underlying the tet2 / ;tet3 / phenotype across differentiation stages. Our results revealed phenotypes in developing tet2 / ;tet3 / retinae including delayed specification of several retinal cell types reduced maturity across late stage tet2 / ;tet3 / cones expansions of immature subpopulations of both horizontal and bipolar cells and altered biases of bipolar cell subtype fates at late differentiation stages. Together these data support Tet proteins as regulators of cell specification lineage fate commitment and terminal differentiation. This study enables a better understanding of how Tet proteins impact the differentiation of many unique yet interacting cell types in a cellularly complex neuronal tissue. Overall design: Embryonic and larval eye cells were collected from 36 48 72 and 120 hpf zebrafish retinae from sibling control sibCTL and tet2 / tet3 / retinae. Dissociated cells were analyzed via scRNAseq,,pubmed:39713311;pubmed:40140485,,36hpf dmut replicate 2,GSM8666793,,source name:eye|tissue:eye|age:36|genotype:tet2 / tet3 / |geo loc name:missing|collection date:missing,36hpf dmut replicate 2,Generation of base count matrices were generated using cellRangerversion 6.1.2. Low quality cells were removed and ambient RNA correction was performed. post ambient RNA correction normalized logcounts were calcluated Assembly: Danio rerio.GRCz11.106 Supplementary files format and content: matrix files,eye,,Whole sibCTL and tet2 / tet3 / eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment in 2 separate wells sibCTL and tet2 / tet3 / dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS. Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads and PCR amplified. post PCR amplification cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,tissue:eye|age:36|genotype:tet2 / tet3 / ,GSM8666793,GSM8666793: 36hpf dmut replicate 2; Danio rerio; RNA Seq,GSM8666793 r1,GSM8666793,1,Whole sibCTL and tet2 / tet3 / eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment in 2 separate wells sibCTL and tet2 / tet3 / dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS. Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads and PCR amplified. post PCR amplification cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP549631,,,36-2_DMUT_Library_GRO2554A2_S5_L001_R1_001.fastq.gz 36-2_DMUT_Library_GRO2554A2_S5_L001_R2_001.fastq.gz,fastq fastq,78416374900.0,664545550.0,GSM8666793 r1,0:28 1:90,A:21577013150;C:17939516025;G:19237904562;T:19657905130;N:4036033,28,90,,,21577013150,17939516025,19237904562,19657905130,4036033,SRX26985240,SRS23451898,SRA2028272,"Gross, Ophthalmology, University of Pittsburgh","Gross, Ophthalmology, University of Pittsburgh",,,,,,,,,,,,T,B,sc-like readlen,illumina,novaseq_era,3prime,poly_a,unknown,sc,single_cell_droplet,10x,,United States,2024-12-05,Multi-stage,Multi-stage,Eye,Sensory System 34265,SRR31620790,SRX26985239,SRS23451897,SRP549631,PRJNA1194578,Tet proteins regulate differentiation potential across retinal cell types,GSE283588,Transcriptome Analysis,Tet enzymes are epigenetic modifiers that impact gene expression via 5mC to 5hmC oxidation; 5hmC generation is known to induce chromatin remodeling and promote transcriptional accessibility and therefore gene expression. Previous work demonstrated the requirement for Tet and 5hmC in early retinogenesis–revealing that Tet nulltet2 / ;tet3 / zebrafish exhibit extensive retinal defects. Here we leveraged scRNAseq technologies to better understand cell type specific deficits and molecular signatures underlying the tet2 / ;tet3 / phenotype across differentiation stages. Our results revealed phenotypes in developing tet2 / ;tet3 / retinae including delayed specification of several retinal cell types reduced maturity across late stage tet2 / ;tet3 / cones expansions of immature subpopulations of both horizontal and bipolar cells and altered biases of bipolar cell subtype fates at late differentiation stages. Together these data support Tet proteins as regulators of cell specification lineage fate commitment and terminal differentiation. This study enables a better understanding of how Tet proteins impact the differentiation of many unique yet interacting cell types in a cellularly complex neuronal tissue. Overall design: Embryonic and larval eye cells were collected from 36 48 72 and 120 hpf zebrafish retinae from sibling control sibCTL and tet2 / tet3 / retinae. Dissociated cells were analyzed via scRNAseq,,pubmed:39713311;pubmed:40140485,,36hpf dmut replicate 1,GSM8666792,,source name:eye|tissue:eye|age:36|genotype:tet2 / tet3 / |geo loc name:missing|collection date:missing,36hpf dmut replicate 1,Generation of base count matrices were generated using cellRangerversion 6.1.2. Low quality cells were removed and ambient RNA correction was performed. post ambient RNA correction normalized logcounts were calcluated Assembly: Danio rerio.GRCz11.106 Supplementary files format and content: matrix files,eye,,Whole sibCTL and tet2 / tet3 / eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment in 2 separate wells sibCTL and tet2 / tet3 / dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS. Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads and PCR amplified. post PCR amplification cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,tissue:eye|age:36|genotype:tet2 / tet3 / ,GSM8666792,GSM8666792: 36hpf dmut replicate 1; Danio rerio; RNA Seq,GSM8666792 r1,GSM8666792,1,Whole sibCTL and tet2 / tet3 / eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment in 2 separate wells sibCTL and tet2 / tet3 / dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS. Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads and PCR amplified. post PCR amplification cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP549631,,,36_1DMUT_LIBRARY_GRO2455A6_S8_L001_R1_001.fastq.gz 36_1DMUT_LIBRARY_GRO2455A6_S8_L001_R2_001.fastq.gz,fastq fastq,59960371084.0,508138738.0,GSM8666792 r1,0:28 1:90,A:16529389286;C:13737763948;G:14793638663;T:14896546895;N:3032292,28,90,,,16529389286,13737763948,14793638663,14896546895,3032292,SRX26985239,SRS23451897,SRA2028272,"Gross, Ophthalmology, University of Pittsburgh","Gross, Ophthalmology, University of Pittsburgh",,,,,,,,,,,,T,B,sc-like readlen,illumina,novaseq_era,3prime,poly_a,unknown,sc,single_cell_droplet,10x,,United States,2024-12-05,Multi-stage,Multi-stage,Eye,Sensory System 34266,SRR31620791,SRX26985238,SRS23451896,SRP549631,PRJNA1194578,Tet proteins regulate differentiation potential across retinal cell types,GSE283588,Transcriptome Analysis,Tet enzymes are epigenetic modifiers that impact gene expression via 5mC to 5hmC oxidation; 5hmC generation is known to induce chromatin remodeling and promote transcriptional accessibility and therefore gene expression. Previous work demonstrated the requirement for Tet and 5hmC in early retinogenesis–revealing that Tet nulltet2 / ;tet3 / zebrafish exhibit extensive retinal defects. Here we leveraged scRNAseq technologies to better understand cell type specific deficits and molecular signatures underlying the tet2 / ;tet3 / phenotype across differentiation stages. Our results revealed phenotypes in developing tet2 / ;tet3 / retinae including delayed specification of several retinal cell types reduced maturity across late stage tet2 / ;tet3 / cones expansions of immature subpopulations of both horizontal and bipolar cells and altered biases of bipolar cell subtype fates at late differentiation stages. Together these data support Tet proteins as regulators of cell specification lineage fate commitment and terminal differentiation. This study enables a better understanding of how Tet proteins impact the differentiation of many unique yet interacting cell types in a cellularly complex neuronal tissue. Overall design: Embryonic and larval eye cells were collected from 36 48 72 and 120 hpf zebrafish retinae from sibling control sibCTL and tet2 / tet3 / retinae. Dissociated cells were analyzed via scRNAseq,,pubmed:39713311;pubmed:40140485,,36hpf sibCTL replicate 2,GSM8666791,,source name:eye|tissue:eye|age:36|genotype:sibCTL|geo loc name:missing|collection date:missing,36hpf sibCTL replicate 2,Generation of base count matrices were generated using cellRangerversion 6.1.2. Low quality cells were removed and ambient RNA correction was performed. post ambient RNA correction normalized logcounts were calcluated Assembly: Danio rerio.GRCz11.106 Supplementary files format and content: matrix files,eye,,Whole sibCTL and tet2 / tet3 / eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment in 2 separate wells sibCTL and tet2 / tet3 / dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS. Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads and PCR amplified. post PCR amplification cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,tissue:eye|age:36|genotype:sibCTL,GSM8666791,GSM8666791: 36hpf sibCTL replicate 2; Danio rerio; RNA Seq,GSM8666791 r1,GSM8666791,1,Whole sibCTL and tet2 / tet3 / eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment in 2 separate wells sibCTL and tet2 / tet3 / dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS. Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads and PCR amplified. post PCR amplification cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP549631,,,36-2_CTL_Library_GRO2554A1_S1_L001_R1_001.fastq.gz 36-2_CTL_Library_GRO2554A1_S1_L001_R2_001.fastq.gz,fastq fastq,75169575046.0,637030297.0,GSM8666791 r1,0:28 1:90,A:20921408123;C:16872043260;G:18114737773;T:19257530991;N:3854899,28,90,,,20921408123,16872043260,18114737773,19257530991,3854899,SRX26985238,SRS23451896,SRA2028272,"Gross, Ophthalmology, University of Pittsburgh","Gross, Ophthalmology, University of Pittsburgh",,,,,,,,,,,,T,B,sc-like readlen,illumina,novaseq_era,3prime,poly_a,unknown,sc,single_cell_droplet,10x,,United States,2024-12-05,Multi-stage,Multi-stage,Eye,Sensory System 34267,SRR31620792,SRX26985237,SRS23451894,SRP549631,PRJNA1194578,Tet proteins regulate differentiation potential across retinal cell types,GSE283588,Transcriptome Analysis,Tet enzymes are epigenetic modifiers that impact gene expression via 5mC to 5hmC oxidation; 5hmC generation is known to induce chromatin remodeling and promote transcriptional accessibility and therefore gene expression. Previous work demonstrated the requirement for Tet and 5hmC in early retinogenesis–revealing that Tet nulltet2 / ;tet3 / zebrafish exhibit extensive retinal defects. Here we leveraged scRNAseq technologies to better understand cell type specific deficits and molecular signatures underlying the tet2 / ;tet3 / phenotype across differentiation stages. Our results revealed phenotypes in developing tet2 / ;tet3 / retinae including delayed specification of several retinal cell types reduced maturity across late stage tet2 / ;tet3 / cones expansions of immature subpopulations of both horizontal and bipolar cells and altered biases of bipolar cell subtype fates at late differentiation stages. Together these data support Tet proteins as regulators of cell specification lineage fate commitment and terminal differentiation. This study enables a better understanding of how Tet proteins impact the differentiation of many unique yet interacting cell types in a cellularly complex neuronal tissue. Overall design: Embryonic and larval eye cells were collected from 36 48 72 and 120 hpf zebrafish retinae from sibling control sibCTL and tet2 / tet3 / retinae. Dissociated cells were analyzed via scRNAseq,,pubmed:39713311;pubmed:40140485,,36hpf sibCTL replicate 1,GSM8666790,,source name:eye|tissue:eye|age:36|genotype:sibCTL|geo loc name:missing|collection date:missing,36hpf sibCTL replicate 1,Generation of base count matrices were generated using cellRangerversion 6.1.2. Low quality cells were removed and ambient RNA correction was performed. post ambient RNA correction normalized logcounts were calcluated Assembly: Danio rerio.GRCz11.106 Supplementary files format and content: matrix files,eye,,Whole sibCTL and tet2 / tet3 / eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment in 2 separate wells sibCTL and tet2 / tet3 / dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS. Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads and PCR amplified. post PCR amplification cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,tissue:eye|age:36|genotype:sibCTL,GSM8666790,GSM8666790: 36hpf sibCTL replicate 1; Danio rerio; RNA Seq,GSM8666790 r1,GSM8666790,1,Whole sibCTL and tet2 / tet3 / eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment in 2 separate wells sibCTL and tet2 / tet3 / dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS. Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads and PCR amplified. post PCR amplification cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP549631,,,36_1CTL_LIBRARY_GRO2455A5_S4_L001_R1_001.fastq.gz 36_1CTL_LIBRARY_GRO2455A5_S4_L001_R2_001.fastq.gz,fastq fastq,70127226422.0,594298529.0,GSM8666790 r1,0:28 1:90,A:19313304121;C:16059894416;G:17310188720;T:17440239265;N:3599900,28,90,,,19313304121,16059894416,17310188720,17440239265,3599900,SRX26985237,SRS23451894,SRA2028272,"Gross, Ophthalmology, University of Pittsburgh","Gross, Ophthalmology, University of Pittsburgh",,,,,,,,,,,,T,B,sc-like readlen,illumina,novaseq_era,3prime,poly_a,unknown,sc,single_cell_droplet,10x,,United States,2024-12-05,Multi-stage,Multi-stage,Eye,Sensory System 34268,SRR31620793,SRX26985236,SRS23451895,SRP549631,PRJNA1194578,Tet proteins regulate differentiation potential across retinal cell types,GSE283588,Transcriptome Analysis,Tet enzymes are epigenetic modifiers that impact gene expression via 5mC to 5hmC oxidation; 5hmC generation is known to induce chromatin remodeling and promote transcriptional accessibility and therefore gene expression. Previous work demonstrated the requirement for Tet and 5hmC in early retinogenesis–revealing that Tet nulltet2 / ;tet3 / zebrafish exhibit extensive retinal defects. Here we leveraged scRNAseq technologies to better understand cell type specific deficits and molecular signatures underlying the tet2 / ;tet3 / phenotype across differentiation stages. Our results revealed phenotypes in developing tet2 / ;tet3 / retinae including delayed specification of several retinal cell types reduced maturity across late stage tet2 / ;tet3 / cones expansions of immature subpopulations of both horizontal and bipolar cells and altered biases of bipolar cell subtype fates at late differentiation stages. Together these data support Tet proteins as regulators of cell specification lineage fate commitment and terminal differentiation. This study enables a better understanding of how Tet proteins impact the differentiation of many unique yet interacting cell types in a cellularly complex neuronal tissue. Overall design: Embryonic and larval eye cells were collected from 36 48 72 and 120 hpf zebrafish retinae from sibling control sibCTL and tet2 / tet3 / retinae. Dissociated cells were analyzed via scRNAseq,,pubmed:39713311;pubmed:40140485,,48hpf dmut replicate 2,GSM8666789,,source name:eye|tissue:eye|age:48|genotype:tet2 / tet3 / |geo loc name:missing|collection date:missing,48hpf dmut replicate 2,Generation of base count matrices were generated using cellRangerversion 6.1.2. Low quality cells were removed and ambient RNA correction was performed. post ambient RNA correction normalized logcounts were calcluated Assembly: Danio rerio.GRCz11.106 Supplementary files format and content: matrix files,eye,,Whole sibCTL and tet2 / tet3 / eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment in 2 separate wells sibCTL and tet2 / tet3 / dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS. Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads and PCR amplified. post PCR amplification cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,tissue:eye|age:48|genotype:tet2 / tet3 / ,GSM8666789,GSM8666789: 48hpf dmut replicate 2; Danio rerio; RNA Seq,GSM8666789 r1,GSM8666789,1,Whole sibCTL and tet2 / tet3 / eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment in 2 separate wells sibCTL and tet2 / tet3 / dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS. Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads and PCR amplified. post PCR amplification cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP549631,,,D48_2_GRO1775A18_S13_L001_R1_001.fastq.gz D48_2_GRO1775A18_S13_L001_R2_001.fastq.gz,fastq fastq,49713593756.0,421301642.0,GSM8666789 r1,0:28 1:90,A:13764945916;C:11293471135;G:11713999638;T:12938678190;N:2498877,28,90,,,13764945916,11293471135,11713999638,12938678190,2498877,SRX26985236,SRS23451895,SRA2028272,"Gross, Ophthalmology, University of Pittsburgh","Gross, Ophthalmology, University of Pittsburgh",,,,,,,,,,,,T,B,sc-like readlen,illumina,novaseq_era,3prime,poly_a,unknown,sc,single_cell_droplet,10x,,United States,2024-12-05,Multi-stage,Multi-stage,Eye,Sensory System 34269,SRR31620794,SRX26985235,SRS23451893,SRP549631,PRJNA1194578,Tet proteins regulate differentiation potential across retinal cell types,GSE283588,Transcriptome Analysis,Tet enzymes are epigenetic modifiers that impact gene expression via 5mC to 5hmC oxidation; 5hmC generation is known to induce chromatin remodeling and promote transcriptional accessibility and therefore gene expression. Previous work demonstrated the requirement for Tet and 5hmC in early retinogenesis–revealing that Tet nulltet2 / ;tet3 / zebrafish exhibit extensive retinal defects. Here we leveraged scRNAseq technologies to better understand cell type specific deficits and molecular signatures underlying the tet2 / ;tet3 / phenotype across differentiation stages. Our results revealed phenotypes in developing tet2 / ;tet3 / retinae including delayed specification of several retinal cell types reduced maturity across late stage tet2 / ;tet3 / cones expansions of immature subpopulations of both horizontal and bipolar cells and altered biases of bipolar cell subtype fates at late differentiation stages. Together these data support Tet proteins as regulators of cell specification lineage fate commitment and terminal differentiation. This study enables a better understanding of how Tet proteins impact the differentiation of many unique yet interacting cell types in a cellularly complex neuronal tissue. Overall design: Embryonic and larval eye cells were collected from 36 48 72 and 120 hpf zebrafish retinae from sibling control sibCTL and tet2 / tet3 / retinae. Dissociated cells were analyzed via scRNAseq,,pubmed:39713311;pubmed:40140485,,48hpf dmut replicate 1,GSM8666788,,source name:eye|tissue:eye|age:48|genotype:tet2 / tet3 / |geo loc name:missing|collection date:missing,48hpf dmut replicate 1,Generation of base count matrices were generated using cellRangerversion 6.1.2. Low quality cells were removed and ambient RNA correction was performed. post ambient RNA correction normalized logcounts were calcluated Assembly: Danio rerio.GRCz11.106 Supplementary files format and content: matrix files,eye,,Whole sibCTL and tet2 / tet3 / eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment in 2 separate wells sibCTL and tet2 / tet3 / dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS. Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads and PCR amplified. post PCR amplification cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,tissue:eye|age:48|genotype:tet2 / tet3 / ,GSM8666788,GSM8666788: 48hpf dmut replicate 1; Danio rerio; RNA Seq,GSM8666788 r1,GSM8666788,1,Whole sibCTL and tet2 / tet3 / eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment in 2 separate wells sibCTL and tet2 / tet3 / dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS. Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads and PCR amplified. post PCR amplification cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP549631,,,D48_1_GRO1775A16_S6_L001_R1_001.fastq.gz D48_1_GRO1775A16_S6_L001_R2_001.fastq.gz,fastq fastq,61017340862.0,517096109.0,GSM8666788 r1,0:28 1:90,A:16897864299;C:13758942283;G:14653497314;T:15703925956;N:3111010,28,90,,,16897864299,13758942283,14653497314,15703925956,3111010,SRX26985235,SRS23451893,SRA2028272,"Gross, Ophthalmology, University of Pittsburgh","Gross, Ophthalmology, University of Pittsburgh",,,,,,,,,,,,T,B,sc-like readlen,illumina,novaseq_era,3prime,poly_a,unknown,sc,single_cell_droplet,10x,,United States,2024-12-05,Multi-stage,Multi-stage,Eye,Sensory System 34270,SRR31620795,SRX26985234,SRS23451891,SRP549631,PRJNA1194578,Tet proteins regulate differentiation potential across retinal cell types,GSE283588,Transcriptome Analysis,Tet enzymes are epigenetic modifiers that impact gene expression via 5mC to 5hmC oxidation; 5hmC generation is known to induce chromatin remodeling and promote transcriptional accessibility and therefore gene expression. Previous work demonstrated the requirement for Tet and 5hmC in early retinogenesis–revealing that Tet nulltet2 / ;tet3 / zebrafish exhibit extensive retinal defects. Here we leveraged scRNAseq technologies to better understand cell type specific deficits and molecular signatures underlying the tet2 / ;tet3 / phenotype across differentiation stages. Our results revealed phenotypes in developing tet2 / ;tet3 / retinae including delayed specification of several retinal cell types reduced maturity across late stage tet2 / ;tet3 / cones expansions of immature subpopulations of both horizontal and bipolar cells and altered biases of bipolar cell subtype fates at late differentiation stages. Together these data support Tet proteins as regulators of cell specification lineage fate commitment and terminal differentiation. This study enables a better understanding of how Tet proteins impact the differentiation of many unique yet interacting cell types in a cellularly complex neuronal tissue. Overall design: Embryonic and larval eye cells were collected from 36 48 72 and 120 hpf zebrafish retinae from sibling control sibCTL and tet2 / tet3 / retinae. Dissociated cells were analyzed via scRNAseq,,pubmed:39713311;pubmed:40140485,,48hpf sibCTL replicate 2,GSM8666787,,source name:eye|tissue:eye|age:48|genotype:sibCTL|geo loc name:missing|collection date:missing,48hpf sibCTL replicate 2,Generation of base count matrices were generated using cellRangerversion 6.1.2. Low quality cells were removed and ambient RNA correction was performed. post ambient RNA correction normalized logcounts were calcluated Assembly: Danio rerio.GRCz11.106 Supplementary files format and content: matrix files,eye,,Whole sibCTL and tet2 / tet3 / eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment in 2 separate wells sibCTL and tet2 / tet3 / dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS. Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads and PCR amplified. post PCR amplification cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,tissue:eye|age:48|genotype:sibCTL,GSM8666787,GSM8666787: 48hpf sibCTL replicate 2; Danio rerio; RNA Seq,GSM8666787 r1,GSM8666787,1,Whole sibCTL and tet2 / tet3 / eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment in 2 separate wells sibCTL and tet2 / tet3 / dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS. Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads and PCR amplified. post PCR amplification cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP549631,,,C48_2_GRO1775A17_S10_L001_R1_001.fastq.gz C48_2_GRO1775A17_S10_L001_R2_001.fastq.gz,fastq fastq,41617041928.0,352686796.0,GSM8666787 r1,0:28 1:90,A:11784184401;C:9157967637;G:9647105654;T:11025644618;N:2139618,28,90,,,11784184401,9157967637,9647105654,11025644618,2139618,SRX26985234,SRS23451891,SRA2028272,"Gross, Ophthalmology, University of Pittsburgh","Gross, Ophthalmology, University of Pittsburgh",,,,,,,,,,,,T,B,sc-like readlen,illumina,novaseq_era,3prime,poly_a,unknown,sc,single_cell_droplet,10x,,United States,2024-12-05,Multi-stage,Multi-stage,Eye,Sensory System 34271,SRR31620796,SRX26985233,SRS23451892,SRP549631,PRJNA1194578,Tet proteins regulate differentiation potential across retinal cell types,GSE283588,Transcriptome Analysis,Tet enzymes are epigenetic modifiers that impact gene expression via 5mC to 5hmC oxidation; 5hmC generation is known to induce chromatin remodeling and promote transcriptional accessibility and therefore gene expression. Previous work demonstrated the requirement for Tet and 5hmC in early retinogenesis–revealing that Tet nulltet2 / ;tet3 / zebrafish exhibit extensive retinal defects. Here we leveraged scRNAseq technologies to better understand cell type specific deficits and molecular signatures underlying the tet2 / ;tet3 / phenotype across differentiation stages. Our results revealed phenotypes in developing tet2 / ;tet3 / retinae including delayed specification of several retinal cell types reduced maturity across late stage tet2 / ;tet3 / cones expansions of immature subpopulations of both horizontal and bipolar cells and altered biases of bipolar cell subtype fates at late differentiation stages. Together these data support Tet proteins as regulators of cell specification lineage fate commitment and terminal differentiation. This study enables a better understanding of how Tet proteins impact the differentiation of many unique yet interacting cell types in a cellularly complex neuronal tissue. Overall design: Embryonic and larval eye cells were collected from 36 48 72 and 120 hpf zebrafish retinae from sibling control sibCTL and tet2 / tet3 / retinae. Dissociated cells were analyzed via scRNAseq,,pubmed:39713311;pubmed:40140485,,48hpf sibCTL replicate 1,GSM8666786,,source name:eye|tissue:eye|age:48|genotype:sibCTL|geo loc name:missing|collection date:missing,48hpf sibCTL replicate 1,Generation of base count matrices were generated using cellRangerversion 6.1.2. Low quality cells were removed and ambient RNA correction was performed. post ambient RNA correction normalized logcounts were calcluated Assembly: Danio rerio.GRCz11.106 Supplementary files format and content: matrix files,eye,,Whole sibCTL and tet2 / tet3 / eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment in 2 separate wells sibCTL and tet2 / tet3 / dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS. Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads and PCR amplified. post PCR amplification cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,tissue:eye|age:48|genotype:sibCTL,GSM8666786,GSM8666786: 48hpf sibCTL replicate 1; Danio rerio; RNA Seq,GSM8666786 r1,GSM8666786,1,Whole sibCTL and tet2 / tet3 / eyes were extracted from zebrafish embryos and larvae and were dissociated using either papain or trypsin with genotype and timepoint specific protocols utilized Library was generated using manaufacturer's instructions scRNAseq v2 three prime Paired End. For each experiment in 2 separate wells sibCTL and tet2 / tet3 / dissociated cells were resuspended in 10x mastermix and loaded with gel beads and partitioning oil to generate GEMS. Each GEM contained sufficient priming material to enable cDNA amplification of poly adenylated mRNA molecules with cell specific10x barcode and transcript specificUMI tags. Pooled cDNA was then cleaned up with silane beads and PCR amplified. post PCR amplification cDNAs were size selected using SPRIselect reagent. During library construction P5 and P7 ends were added for sequencing.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP549631,,,C48_1_GRO1775A15_S2_L001_R1_001.fastq.gz C48_1_GRO1775A15_S2_L001_R2_001.fastq.gz,fastq fastq,54112599182.0,458581349.0,GSM8666786 r1,0:28 1:90,A:15198773029;C:11981463118;G:12618365415;T:14311218195;N:2779425,28,90,,,15198773029,11981463118,12618365415,14311218195,2779425,SRX26985233,SRS23451892,SRA2028272,"Gross, Ophthalmology, University of Pittsburgh","Gross, Ophthalmology, University of Pittsburgh",,,,,,,,,,,,T,B,sc-like readlen,illumina,novaseq_era,3prime,poly_a,unknown,sc,single_cell_droplet,10x,,United States,2024-12-05,Multi-stage,Multi-stage,Eye,Sensory 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 59859,SRR12054798,SRX8583217,SRS6874834,SRP268010,PRJNA640615,Molecular classification of zebrafish retinal ganglion cells links genes to behavior,GSE152842,Transcriptome Analysis,Retinal ganglion cells RGCs form an array of feature detectors which transmit visual information through segregated channels to central brain regions. Characterizing RGC diversity is required to understand the logic of this functional parcellation. Using single cell transcriptomics we systematically classified RGCs in adult and larval zebrafish thereby identifying marker genes for >30 stable and several transient cell types cell type specific markers and their maturational changes. We used this catalog to engineer transgenic driverreporter lines enabling experimental genetic access to specific RGC types. Strikingly expression of a few transcription factors often predicts dendrite morphological features in particular and axonal projections to specific tectal layers and extratectal targets. Moreover iIn vivo calcium imaging revealed that molecularly defined RGCs exhibit highly specific functional tuningphysiological properties. Finally chemogenetic ablation of eomesa+ RGCs which comprise three melanopsin expressing types with a specific projections to thalamus pretectum and one deep tectal layerpattern selectively impaired a visual behavior phototaxis. Our study establishes a framework and provides a resource for systematically studying the functional architecture of the visual system. Overall design: RGCs were labeled using transgenic Tgisl2b:tagRFP zebrafish that express RFP in all RGCs. Retinas from larval 5 dpf and adult 4 month 6 month fish were dissected and sorted using FACS. For the larval dataset 200 manually dissected retinas were dissociated in one experiment and single cell profiles were collected across 3 replicates. For the adult dataset about 20 retinas per batch were dissected and dissociated and droplet RNA sequencing was performed collecting a total of 15 replicates across 5 experiments. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell.,,pubmed:33357413,,Adult Zebrafish 15,GSM4627227,,source name:Zebrafish retina 4 mpf 6 mpf|tissue:retina,Adult Zebrafish 15,Sample demultiplexing for each of 15 adult and 3 larval samples with the mkfastq function from Cell Ranger softwareversion 2.1.0 10X Genomics Sample alignment to the zebrafish reference transcriptome ENSEMBL zv10 release 82 using “cellranger count” with default parameters to obtain a digital gene expression DGE matrix genes x cells summarizing transcript counts. Genome build: ENSEMBL zv10 release 82 Supplementary files format and content: Csv files including gene expression matrix from all larval samples and all adult samples,Zebrafish retina 4 mpf 6 mpf,All zebrafish used were anesthetized in a lethal overdose of tricaine and rapidly euthanized by immersion in ice water for 10 min. Retinas from larval or adult fish were dissected in oxygenated ox Ames and transferred into ox Ames on ice until tissue collection was completed.,Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 larval retinas or 45 minutes adult retinas. To stop the digestion the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence activated cell sorting FACS purification. Non transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within 45 minutes post FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell. 10x Chromium 3’ v2 platform 10X Genomics Pleasanton CA,Adult and larval zebrafish were maintained on a 14:10 hour light:dark cycle at 28°C. Embryos were bred in Danieau’s solution 17 mM NaCl 2 mM KCl 0.12 mM MgSO4 1.8 mM CaNO32 1.5 mM HEPES.,tissue:retina,GSM4627227,GSM4627227: Adult Zebrafish 15; Danio rerio; RNA Seq,GSM4627227,,1,Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 larval retinas or 45 minutes adult retinas. To stop the digestion the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence activated cell sorting FACS purification. Non transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within 45 minutes post FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell. 10x Chromium three prime v2 platform 10X Genomics Pleasanton CA,GEO Accession:GSM4627227,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP268010,,assembly:GRCz10|intentional duplicate,ZebrafishRGC15.bam,10X Genomics bam file,17489597440.0,178465280.0,GSM4627227 r1,0:98,A:5144837324;C:3453657332;G:3688751956;T:5199397147;N:2953681,98,,,,5144837324,3453657332,3688751956,5199397147,2953681,SRX8583217,SRS6874834,SRA1089100,GEO,"Karthik Shekhar, Chemical and Biomolecular Engineering, UC Berkeley",1,0.88384,,0.24019,,0.78979,,0.51275,,98,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-06-19,Multi-stage,Multi-stage,Eye,Sensory System 59860,SRR12054797,SRX8583216,SRS6874833,SRP268010,PRJNA640615,Molecular classification of zebrafish retinal ganglion cells links genes to behavior,GSE152842,Transcriptome Analysis,Retinal ganglion cells RGCs form an array of feature detectors which transmit visual information through segregated channels to central brain regions. Characterizing RGC diversity is required to understand the logic of this functional parcellation. Using single cell transcriptomics we systematically classified RGCs in adult and larval zebrafish thereby identifying marker genes for >30 stable and several transient cell types cell type specific markers and their maturational changes. We used this catalog to engineer transgenic driverreporter lines enabling experimental genetic access to specific RGC types. Strikingly expression of a few transcription factors often predicts dendrite morphological features in particular and axonal projections to specific tectal layers and extratectal targets. Moreover iIn vivo calcium imaging revealed that molecularly defined RGCs exhibit highly specific functional tuningphysiological properties. Finally chemogenetic ablation of eomesa+ RGCs which comprise three melanopsin expressing types with a specific projections to thalamus pretectum and one deep tectal layerpattern selectively impaired a visual behavior phototaxis. Our study establishes a framework and provides a resource for systematically studying the functional architecture of the visual system. Overall design: RGCs were labeled using transgenic Tgisl2b:tagRFP zebrafish that express RFP in all RGCs. Retinas from larval 5 dpf and adult 4 month 6 month fish were dissected and sorted using FACS. For the larval dataset 200 manually dissected retinas were dissociated in one experiment and single cell profiles were collected across 3 replicates. For the adult dataset about 20 retinas per batch were dissected and dissociated and droplet RNA sequencing was performed collecting a total of 15 replicates across 5 experiments. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell.,,pubmed:33357413,,Adult Zebrafish 14,GSM4627226,,source name:Zebrafish retina 4 mpf 6 mpf|tissue:retina,Adult Zebrafish 14,Sample demultiplexing for each of 15 adult and 3 larval samples with the mkfastq function from Cell Ranger softwareversion 2.1.0 10X Genomics Sample alignment to the zebrafish reference transcriptome ENSEMBL zv10 release 82 using “cellranger count” with default parameters to obtain a digital gene expression DGE matrix genes x cells summarizing transcript counts. Genome build: ENSEMBL zv10 release 82 Supplementary files format and content: Csv files including gene expression matrix from all larval samples and all adult samples,Zebrafish retina 4 mpf 6 mpf,All zebrafish used were anesthetized in a lethal overdose of tricaine and rapidly euthanized by immersion in ice water for 10 min. Retinas from larval or adult fish were dissected in oxygenated ox Ames and transferred into ox Ames on ice until tissue collection was completed.,Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 larval retinas or 45 minutes adult retinas. To stop the digestion the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence activated cell sorting FACS purification. Non transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within 45 minutes post FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell. 10x Chromium 3’ v2 platform 10X Genomics Pleasanton CA,Adult and larval zebrafish were maintained on a 14:10 hour light:dark cycle at 28°C. Embryos were bred in Danieau’s solution 17 mM NaCl 2 mM KCl 0.12 mM MgSO4 1.8 mM CaNO32 1.5 mM HEPES.,tissue:retina,GSM4627226,GSM4627226: Adult Zebrafish 14; Danio rerio; RNA Seq,GSM4627226,,1,Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 larval retinas or 45 minutes adult retinas. To stop the digestion the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence activated cell sorting FACS purification. Non transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within 45 minutes post FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell. 10x Chromium three prime v2 platform 10X Genomics Pleasanton CA,GEO Accession:GSM4627226,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP268010,,assembly:GRCz10|intentional duplicate,ZebrafishRGC14.bam,10X Genomics bam file,16163588938.0,164934581.0,GSM4627226 r1,0:98,A:4767424342;C:3189871576;G:3410422503;T:4793151919;N:2718598,98,,,,4767424342,3189871576,3410422503,4793151919,2718598,SRX8583216,SRS6874833,SRA1089100,GEO,"Karthik Shekhar, Chemical and Biomolecular Engineering, UC Berkeley",1,0.88242,,0.24257,,0.78892,,0.49263,,98,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-06-19,Multi-stage,Multi-stage,Eye,Sensory System 59861,SRR12054796,SRX8583215,SRS6874832,SRP268010,PRJNA640615,Molecular classification of zebrafish retinal ganglion cells links genes to behavior,GSE152842,Transcriptome Analysis,Retinal ganglion cells RGCs form an array of feature detectors which transmit visual information through segregated channels to central brain regions. Characterizing RGC diversity is required to understand the logic of this functional parcellation. Using single cell transcriptomics we systematically classified RGCs in adult and larval zebrafish thereby identifying marker genes for >30 stable and several transient cell types cell type specific markers and their maturational changes. We used this catalog to engineer transgenic driverreporter lines enabling experimental genetic access to specific RGC types. Strikingly expression of a few transcription factors often predicts dendrite morphological features in particular and axonal projections to specific tectal layers and extratectal targets. Moreover iIn vivo calcium imaging revealed that molecularly defined RGCs exhibit highly specific functional tuningphysiological properties. Finally chemogenetic ablation of eomesa+ RGCs which comprise three melanopsin expressing types with a specific projections to thalamus pretectum and one deep tectal layerpattern selectively impaired a visual behavior phototaxis. Our study establishes a framework and provides a resource for systematically studying the functional architecture of the visual system. Overall design: RGCs were labeled using transgenic Tgisl2b:tagRFP zebrafish that express RFP in all RGCs. Retinas from larval 5 dpf and adult 4 month 6 month fish were dissected and sorted using FACS. For the larval dataset 200 manually dissected retinas were dissociated in one experiment and single cell profiles were collected across 3 replicates. For the adult dataset about 20 retinas per batch were dissected and dissociated and droplet RNA sequencing was performed collecting a total of 15 replicates across 5 experiments. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell.,,pubmed:33357413,,Adult Zebrafish 13,GSM4627225,,source name:Zebrafish retina 4 mpf 6 mpf|tissue:retina,Adult Zebrafish 13,Sample demultiplexing for each of 15 adult and 3 larval samples with the mkfastq function from Cell Ranger softwareversion 2.1.0 10X Genomics Sample alignment to the zebrafish reference transcriptome ENSEMBL zv10 release 82 using “cellranger count” with default parameters to obtain a digital gene expression DGE matrix genes x cells summarizing transcript counts. Genome build: ENSEMBL zv10 release 82 Supplementary files format and content: Csv files including gene expression matrix from all larval samples and all adult samples,Zebrafish retina 4 mpf 6 mpf,All zebrafish used were anesthetized in a lethal overdose of tricaine and rapidly euthanized by immersion in ice water for 10 min. Retinas from larval or adult fish were dissected in oxygenated ox Ames and transferred into ox Ames on ice until tissue collection was completed.,Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 larval retinas or 45 minutes adult retinas. To stop the digestion the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence activated cell sorting FACS purification. Non transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within 45 minutes post FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell. 10x Chromium 3’ v2 platform 10X Genomics Pleasanton CA,Adult and larval zebrafish were maintained on a 14:10 hour light:dark cycle at 28°C. Embryos were bred in Danieau’s solution 17 mM NaCl 2 mM KCl 0.12 mM MgSO4 1.8 mM CaNO32 1.5 mM HEPES.,tissue:retina,GSM4627225,GSM4627225: Adult Zebrafish 13; Danio rerio; RNA Seq,GSM4627225,,1,Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 larval retinas or 45 minutes adult retinas. To stop the digestion the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence activated cell sorting FACS purification. Non transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within 45 minutes post FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell. 10x Chromium three prime v2 platform 10X Genomics Pleasanton CA,GEO Accession:GSM4627225,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP268010,,assembly:GRCz10|intentional duplicate,ZebrafishRGC13.bam,10X Genomics bam file,14166392450.0,144555025.0,GSM4627225 r1,0:98,A:4156001799;C:2794969042;G:2979050667;T:4233954883;N:2416059,98,,,,4156001799,2794969042,2979050667,4233954883,2416059,SRX8583215,SRS6874832,SRA1089100,GEO,"Karthik Shekhar, Chemical and Biomolecular Engineering, UC Berkeley",1,0.88338,,0.24795,,0.7893,,0.49627,,98,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-06-19,Multi-stage,Multi-stage,Eye,Sensory System 59862,SRR12054795,SRX8583214,SRS6874831,SRP268010,PRJNA640615,Molecular classification of zebrafish retinal ganglion cells links genes to behavior,GSE152842,Transcriptome Analysis,Retinal ganglion cells RGCs form an array of feature detectors which transmit visual information through segregated channels to central brain regions. Characterizing RGC diversity is required to understand the logic of this functional parcellation. Using single cell transcriptomics we systematically classified RGCs in adult and larval zebrafish thereby identifying marker genes for >30 stable and several transient cell types cell type specific markers and their maturational changes. We used this catalog to engineer transgenic driverreporter lines enabling experimental genetic access to specific RGC types. Strikingly expression of a few transcription factors often predicts dendrite morphological features in particular and axonal projections to specific tectal layers and extratectal targets. Moreover iIn vivo calcium imaging revealed that molecularly defined RGCs exhibit highly specific functional tuningphysiological properties. Finally chemogenetic ablation of eomesa+ RGCs which comprise three melanopsin expressing types with a specific projections to thalamus pretectum and one deep tectal layerpattern selectively impaired a visual behavior phototaxis. Our study establishes a framework and provides a resource for systematically studying the functional architecture of the visual system. Overall design: RGCs were labeled using transgenic Tgisl2b:tagRFP zebrafish that express RFP in all RGCs. Retinas from larval 5 dpf and adult 4 month 6 month fish were dissected and sorted using FACS. For the larval dataset 200 manually dissected retinas were dissociated in one experiment and single cell profiles were collected across 3 replicates. For the adult dataset about 20 retinas per batch were dissected and dissociated and droplet RNA sequencing was performed collecting a total of 15 replicates across 5 experiments. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell.,,pubmed:33357413,,Adult Zebrafish 12,GSM4627224,,source name:Zebrafish retina 4 mpf 6 mpf|tissue:retina,Adult Zebrafish 12,Sample demultiplexing for each of 15 adult and 3 larval samples with the mkfastq function from Cell Ranger softwareversion 2.1.0 10X Genomics Sample alignment to the zebrafish reference transcriptome ENSEMBL zv10 release 82 using “cellranger count” with default parameters to obtain a digital gene expression DGE matrix genes x cells summarizing transcript counts. Genome build: ENSEMBL zv10 release 82 Supplementary files format and content: Csv files including gene expression matrix from all larval samples and all adult samples,Zebrafish retina 4 mpf 6 mpf,All zebrafish used were anesthetized in a lethal overdose of tricaine and rapidly euthanized by immersion in ice water for 10 min. Retinas from larval or adult fish were dissected in oxygenated ox Ames and transferred into ox Ames on ice until tissue collection was completed.,Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 larval retinas or 45 minutes adult retinas. To stop the digestion the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence activated cell sorting FACS purification. Non transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within 45 minutes post FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell. 10x Chromium 3’ v2 platform 10X Genomics Pleasanton CA,Adult and larval zebrafish were maintained on a 14:10 hour light:dark cycle at 28°C. Embryos were bred in Danieau’s solution 17 mM NaCl 2 mM KCl 0.12 mM MgSO4 1.8 mM CaNO32 1.5 mM HEPES.,tissue:retina,GSM4627224,GSM4627224: Adult Zebrafish 12; Danio rerio; RNA Seq,GSM4627224,,1,Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 larval retinas or 45 minutes adult retinas. To stop the digestion the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence activated cell sorting FACS purification. Non transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within 45 minutes post FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell. 10x Chromium three prime v2 platform 10X Genomics Pleasanton CA,GEO Accession:GSM4627224,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP268010,,assembly:GRCz10|intentional duplicate,ZebrafishRGC12.bam,10X Genomics bam file,15484230986.0,158002357.0,GSM4627224 r1,0:98,A:4556531984;C:3035955488;G:3233886544;T:4655230637;N:2626333,98,,,,4556531984,3035955488,3233886544,4655230637,2626333,SRX8583214,SRS6874831,SRA1089100,GEO,"Karthik Shekhar, Chemical and Biomolecular Engineering, UC Berkeley",1,0.88248,,0.25141,,0.78782,,0.49785,,98,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-06-19,Multi-stage,Multi-stage,Eye,Sensory System 59863,SRR12054794,SRX8583213,SRS6874830,SRP268010,PRJNA640615,Molecular classification of zebrafish retinal ganglion cells links genes to behavior,GSE152842,Transcriptome Analysis,Retinal ganglion cells RGCs form an array of feature detectors which transmit visual information through segregated channels to central brain regions. Characterizing RGC diversity is required to understand the logic of this functional parcellation. Using single cell transcriptomics we systematically classified RGCs in adult and larval zebrafish thereby identifying marker genes for >30 stable and several transient cell types cell type specific markers and their maturational changes. We used this catalog to engineer transgenic driverreporter lines enabling experimental genetic access to specific RGC types. Strikingly expression of a few transcription factors often predicts dendrite morphological features in particular and axonal projections to specific tectal layers and extratectal targets. Moreover iIn vivo calcium imaging revealed that molecularly defined RGCs exhibit highly specific functional tuningphysiological properties. Finally chemogenetic ablation of eomesa+ RGCs which comprise three melanopsin expressing types with a specific projections to thalamus pretectum and one deep tectal layerpattern selectively impaired a visual behavior phototaxis. Our study establishes a framework and provides a resource for systematically studying the functional architecture of the visual system. Overall design: RGCs were labeled using transgenic Tgisl2b:tagRFP zebrafish that express RFP in all RGCs. Retinas from larval 5 dpf and adult 4 month 6 month fish were dissected and sorted using FACS. For the larval dataset 200 manually dissected retinas were dissociated in one experiment and single cell profiles were collected across 3 replicates. For the adult dataset about 20 retinas per batch were dissected and dissociated and droplet RNA sequencing was performed collecting a total of 15 replicates across 5 experiments. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell.,,pubmed:33357413,,Adult Zebrafish 11,GSM4627223,,source name:Zebrafish retina 4 mpf 6 mpf|tissue:retina,Adult Zebrafish 11,Sample demultiplexing for each of 15 adult and 3 larval samples with the mkfastq function from Cell Ranger softwareversion 2.1.0 10X Genomics Sample alignment to the zebrafish reference transcriptome ENSEMBL zv10 release 82 using “cellranger count” with default parameters to obtain a digital gene expression DGE matrix genes x cells summarizing transcript counts. Genome build: ENSEMBL zv10 release 82 Supplementary files format and content: Csv files including gene expression matrix from all larval samples and all adult samples,Zebrafish retina 4 mpf 6 mpf,All zebrafish used were anesthetized in a lethal overdose of tricaine and rapidly euthanized by immersion in ice water for 10 min. Retinas from larval or adult fish were dissected in oxygenated ox Ames and transferred into ox Ames on ice until tissue collection was completed.,Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 larval retinas or 45 minutes adult retinas. To stop the digestion the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence activated cell sorting FACS purification. Non transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within 45 minutes post FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell. 10x Chromium 3’ v2 platform 10X Genomics Pleasanton CA,Adult and larval zebrafish were maintained on a 14:10 hour light:dark cycle at 28°C. Embryos were bred in Danieau’s solution 17 mM NaCl 2 mM KCl 0.12 mM MgSO4 1.8 mM CaNO32 1.5 mM HEPES.,tissue:retina,GSM4627223,GSM4627223: Adult Zebrafish 11; Danio rerio; RNA Seq,GSM4627223,,1,Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 larval retinas or 45 minutes adult retinas. To stop the digestion the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence activated cell sorting FACS purification. Non transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within 45 minutes post FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell. 10x Chromium three prime v2 platform 10X Genomics Pleasanton CA,GEO Accession:GSM4627223,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP268010,,assembly:GRCz10|intentional duplicate,ZebrafishRGC11.bam,10X Genomics bam file,18012879710.0,183804895.0,GSM4627223 r1,0:98,A:5278772392;C:3561457870;G:3799039640;T:5370568922;N:3040886,98,,,,5278772392,3561457870,3799039640,5370568922,3040886,SRX8583213,SRS6874830,SRA1089100,GEO,"Karthik Shekhar, Chemical and Biomolecular Engineering, UC Berkeley",1,0.88456,,0.25211,,0.7894,,0.50115,,98,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-06-19,Multi-stage,Multi-stage,Eye,Sensory System 59864,SRR12054791,SRX8583210,SRS6874827,SRP268010,PRJNA640615,Molecular classification of zebrafish retinal ganglion cells links genes to behavior,GSE152842,Transcriptome Analysis,Retinal ganglion cells RGCs form an array of feature detectors which transmit visual information through segregated channels to central brain regions. Characterizing RGC diversity is required to understand the logic of this functional parcellation. Using single cell transcriptomics we systematically classified RGCs in adult and larval zebrafish thereby identifying marker genes for >30 stable and several transient cell types cell type specific markers and their maturational changes. We used this catalog to engineer transgenic driverreporter lines enabling experimental genetic access to specific RGC types. Strikingly expression of a few transcription factors often predicts dendrite morphological features in particular and axonal projections to specific tectal layers and extratectal targets. Moreover iIn vivo calcium imaging revealed that molecularly defined RGCs exhibit highly specific functional tuningphysiological properties. Finally chemogenetic ablation of eomesa+ RGCs which comprise three melanopsin expressing types with a specific projections to thalamus pretectum and one deep tectal layerpattern selectively impaired a visual behavior phototaxis. Our study establishes a framework and provides a resource for systematically studying the functional architecture of the visual system. Overall design: RGCs were labeled using transgenic Tgisl2b:tagRFP zebrafish that express RFP in all RGCs. Retinas from larval 5 dpf and adult 4 month 6 month fish were dissected and sorted using FACS. For the larval dataset 200 manually dissected retinas were dissociated in one experiment and single cell profiles were collected across 3 replicates. For the adult dataset about 20 retinas per batch were dissected and dissociated and droplet RNA sequencing was performed collecting a total of 15 replicates across 5 experiments. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell.,,pubmed:33357413,,Adult Zebrafish 8,GSM4627220,,source name:Zebrafish retina 4 mpf 6 mpf|tissue:retina,Adult Zebrafish 8,Sample demultiplexing for each of 15 adult and 3 larval samples with the mkfastq function from Cell Ranger softwareversion 2.1.0 10X Genomics Sample alignment to the zebrafish reference transcriptome ENSEMBL zv10 release 82 using “cellranger count” with default parameters to obtain a digital gene expression DGE matrix genes x cells summarizing transcript counts. Genome build: ENSEMBL zv10 release 82 Supplementary files format and content: Csv files including gene expression matrix from all larval samples and all adult samples,Zebrafish retina 4 mpf 6 mpf,All zebrafish used were anesthetized in a lethal overdose of tricaine and rapidly euthanized by immersion in ice water for 10 min. Retinas from larval or adult fish were dissected in oxygenated ox Ames and transferred into ox Ames on ice until tissue collection was completed.,Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 larval retinas or 45 minutes adult retinas. To stop the digestion the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence activated cell sorting FACS purification. Non transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within 45 minutes post FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell. 10x Chromium 3’ v2 platform 10X Genomics Pleasanton CA,Adult and larval zebrafish were maintained on a 14:10 hour light:dark cycle at 28°C. Embryos were bred in Danieau’s solution 17 mM NaCl 2 mM KCl 0.12 mM MgSO4 1.8 mM CaNO32 1.5 mM HEPES.,tissue:retina,GSM4627220,GSM4627220: Adult Zebrafish 8; Danio rerio; RNA Seq,GSM4627220,,1,Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 larval retinas or 45 minutes adult retinas. To stop the digestion the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence activated cell sorting FACS purification. Non transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within 45 minutes post FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell. 10x Chromium three prime v2 platform 10X Genomics Pleasanton CA,GEO Accession:GSM4627220,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP268010,,assembly:GRCz10|intentional duplicate,ZebrafishRGC8.bam,10X Genomics bam file,14551197976.0,148481612.0,GSM4627220 r1,0:98,A:4325239722;C:2863430626;G:2994943270;T:4365215038;N:2369320,98,,,,4325239722,2863430626,2994943270,4365215038,2369320,SRX8583210,SRS6874827,SRA1089100,GEO,"Karthik Shekhar, Chemical and Biomolecular Engineering, UC Berkeley",1,0.87211,,0.29067,,0.79431,,0.49957,,98,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-06-19,Multi-stage,Multi-stage,Eye,Sensory System 59865,SRR12054790,SRX8583209,SRS6874826,SRP268010,PRJNA640615,Molecular classification of zebrafish retinal ganglion cells links genes to behavior,GSE152842,Transcriptome Analysis,Retinal ganglion cells RGCs form an array of feature detectors which transmit visual information through segregated channels to central brain regions. Characterizing RGC diversity is required to understand the logic of this functional parcellation. Using single cell transcriptomics we systematically classified RGCs in adult and larval zebrafish thereby identifying marker genes for >30 stable and several transient cell types cell type specific markers and their maturational changes. We used this catalog to engineer transgenic driverreporter lines enabling experimental genetic access to specific RGC types. Strikingly expression of a few transcription factors often predicts dendrite morphological features in particular and axonal projections to specific tectal layers and extratectal targets. Moreover iIn vivo calcium imaging revealed that molecularly defined RGCs exhibit highly specific functional tuningphysiological properties. Finally chemogenetic ablation of eomesa+ RGCs which comprise three melanopsin expressing types with a specific projections to thalamus pretectum and one deep tectal layerpattern selectively impaired a visual behavior phototaxis. Our study establishes a framework and provides a resource for systematically studying the functional architecture of the visual system. Overall design: RGCs were labeled using transgenic Tgisl2b:tagRFP zebrafish that express RFP in all RGCs. Retinas from larval 5 dpf and adult 4 month 6 month fish were dissected and sorted using FACS. For the larval dataset 200 manually dissected retinas were dissociated in one experiment and single cell profiles were collected across 3 replicates. For the adult dataset about 20 retinas per batch were dissected and dissociated and droplet RNA sequencing was performed collecting a total of 15 replicates across 5 experiments. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell.,,pubmed:33357413,,Adult Zebrafish 7,GSM4627219,,source name:Zebrafish retina 4 mpf 6 mpf|tissue:retina,Adult Zebrafish 7,Sample demultiplexing for each of 15 adult and 3 larval samples with the mkfastq function from Cell Ranger softwareversion 2.1.0 10X Genomics Sample alignment to the zebrafish reference transcriptome ENSEMBL zv10 release 82 using “cellranger count” with default parameters to obtain a digital gene expression DGE matrix genes x cells summarizing transcript counts. Genome build: ENSEMBL zv10 release 82 Supplementary files format and content: Csv files including gene expression matrix from all larval samples and all adult samples,Zebrafish retina 4 mpf 6 mpf,All zebrafish used were anesthetized in a lethal overdose of tricaine and rapidly euthanized by immersion in ice water for 10 min. Retinas from larval or adult fish were dissected in oxygenated ox Ames and transferred into ox Ames on ice until tissue collection was completed.,Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 larval retinas or 45 minutes adult retinas. To stop the digestion the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence activated cell sorting FACS purification. Non transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within 45 minutes post FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell. 10x Chromium 3’ v2 platform 10X Genomics Pleasanton CA,Adult and larval zebrafish were maintained on a 14:10 hour light:dark cycle at 28°C. Embryos were bred in Danieau’s solution 17 mM NaCl 2 mM KCl 0.12 mM MgSO4 1.8 mM CaNO32 1.5 mM HEPES.,tissue:retina,GSM4627219,GSM4627219: Adult Zebrafish 7; Danio rerio; RNA Seq,GSM4627219,,1,Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 larval retinas or 45 minutes adult retinas. To stop the digestion the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence activated cell sorting FACS purification. Non transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within 45 minutes post FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell. 10x Chromium three prime v2 platform 10X Genomics Pleasanton CA,GEO Accession:GSM4627219,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP268010,,assembly:GRCz10|intentional duplicate,ZebrafishRGC7.bam,10X Genomics bam file,12894390180.0,131575410.0,GSM4627219 r1,0:98,A:3809964234;C:2548974567;G:2667179504;T:3866166531;N:2105344,98,,,,3809964234,2548974567,2667179504,3866166531,2105344,SRX8583209,SRS6874826,SRA1089100,GEO,"Karthik Shekhar, Chemical and Biomolecular Engineering, UC Berkeley",1,0.87565,,0.28214,,0.78918,,0.51085,,98,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-06-19,Multi-stage,Multi-stage,Eye,Sensory System 59866,SRR12054789,SRX8583208,SRS6874825,SRP268010,PRJNA640615,Molecular classification of zebrafish retinal ganglion cells links genes to behavior,GSE152842,Transcriptome Analysis,Retinal ganglion cells RGCs form an array of feature detectors which transmit visual information through segregated channels to central brain regions. Characterizing RGC diversity is required to understand the logic of this functional parcellation. Using single cell transcriptomics we systematically classified RGCs in adult and larval zebrafish thereby identifying marker genes for >30 stable and several transient cell types cell type specific markers and their maturational changes. We used this catalog to engineer transgenic driverreporter lines enabling experimental genetic access to specific RGC types. Strikingly expression of a few transcription factors often predicts dendrite morphological features in particular and axonal projections to specific tectal layers and extratectal targets. Moreover iIn vivo calcium imaging revealed that molecularly defined RGCs exhibit highly specific functional tuningphysiological properties. Finally chemogenetic ablation of eomesa+ RGCs which comprise three melanopsin expressing types with a specific projections to thalamus pretectum and one deep tectal layerpattern selectively impaired a visual behavior phototaxis. Our study establishes a framework and provides a resource for systematically studying the functional architecture of the visual system. Overall design: RGCs were labeled using transgenic Tgisl2b:tagRFP zebrafish that express RFP in all RGCs. Retinas from larval 5 dpf and adult 4 month 6 month fish were dissected and sorted using FACS. For the larval dataset 200 manually dissected retinas were dissociated in one experiment and single cell profiles were collected across 3 replicates. For the adult dataset about 20 retinas per batch were dissected and dissociated and droplet RNA sequencing was performed collecting a total of 15 replicates across 5 experiments. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell.,,pubmed:33357413,,Adult Zebrafish 6,GSM4627218,,source name:Zebrafish retina 4 mpf 6 mpf|tissue:retina,Adult Zebrafish 6,Sample demultiplexing for each of 15 adult and 3 larval samples with the mkfastq function from Cell Ranger softwareversion 2.1.0 10X Genomics Sample alignment to the zebrafish reference transcriptome ENSEMBL zv10 release 82 using “cellranger count” with default parameters to obtain a digital gene expression DGE matrix genes x cells summarizing transcript counts. Genome build: ENSEMBL zv10 release 82 Supplementary files format and content: Csv files including gene expression matrix from all larval samples and all adult samples,Zebrafish retina 4 mpf 6 mpf,All zebrafish used were anesthetized in a lethal overdose of tricaine and rapidly euthanized by immersion in ice water for 10 min. Retinas from larval or adult fish were dissected in oxygenated ox Ames and transferred into ox Ames on ice until tissue collection was completed.,Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 larval retinas or 45 minutes adult retinas. To stop the digestion the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence activated cell sorting FACS purification. Non transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within 45 minutes post FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell. 10x Chromium 3’ v2 platform 10X Genomics Pleasanton CA,Adult and larval zebrafish were maintained on a 14:10 hour light:dark cycle at 28°C. Embryos were bred in Danieau’s solution 17 mM NaCl 2 mM KCl 0.12 mM MgSO4 1.8 mM CaNO32 1.5 mM HEPES.,tissue:retina,GSM4627218,GSM4627218: Adult Zebrafish 6; Danio rerio; RNA Seq,GSM4627218,,1,Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 larval retinas or 45 minutes adult retinas. To stop the digestion the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence activated cell sorting FACS purification. Non transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within 45 minutes post FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell. 10x Chromium three prime v2 platform 10X Genomics Pleasanton CA,GEO Accession:GSM4627218,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP268010,,assembly:GRCz10|intentional duplicate,ZebrafishRGC6.bam,10X Genomics bam file,11333584262.0,115648819.0,GSM4627218 r1,0:98,A:3357618948;C:2223130462;G:2352697999;T:3398318533;N:1818320,98,,,,3357618948,2223130462,2352697999,3398318533,1818320,SRX8583208,SRS6874825,SRA1089100,GEO,"Karthik Shekhar, Chemical and Biomolecular Engineering, UC Berkeley",1,0.87718,,0.26282,,0.7838,,0.50701,,98,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-06-19,Multi-stage,Multi-stage,Eye,Sensory System 59867,SRR12054786,SRX8583205,SRS6874822,SRP268010,PRJNA640615,Molecular classification of zebrafish retinal ganglion cells links genes to behavior,GSE152842,Transcriptome Analysis,Retinal ganglion cells RGCs form an array of feature detectors which transmit visual information through segregated channels to central brain regions. Characterizing RGC diversity is required to understand the logic of this functional parcellation. Using single cell transcriptomics we systematically classified RGCs in adult and larval zebrafish thereby identifying marker genes for >30 stable and several transient cell types cell type specific markers and their maturational changes. We used this catalog to engineer transgenic driverreporter lines enabling experimental genetic access to specific RGC types. Strikingly expression of a few transcription factors often predicts dendrite morphological features in particular and axonal projections to specific tectal layers and extratectal targets. Moreover iIn vivo calcium imaging revealed that molecularly defined RGCs exhibit highly specific functional tuningphysiological properties. Finally chemogenetic ablation of eomesa+ RGCs which comprise three melanopsin expressing types with a specific projections to thalamus pretectum and one deep tectal layerpattern selectively impaired a visual behavior phototaxis. Our study establishes a framework and provides a resource for systematically studying the functional architecture of the visual system. Overall design: RGCs were labeled using transgenic Tgisl2b:tagRFP zebrafish that express RFP in all RGCs. Retinas from larval 5 dpf and adult 4 month 6 month fish were dissected and sorted using FACS. For the larval dataset 200 manually dissected retinas were dissociated in one experiment and single cell profiles were collected across 3 replicates. For the adult dataset about 20 retinas per batch were dissected and dissociated and droplet RNA sequencing was performed collecting a total of 15 replicates across 5 experiments. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell.,,pubmed:33357413,,Adult Zebrafish 3,GSM4627215,,source name:Zebrafish retina 4 mpf 6 mpf|tissue:retina,Adult Zebrafish 3,Sample demultiplexing for each of 15 adult and 3 larval samples with the mkfastq function from Cell Ranger softwareversion 2.1.0 10X Genomics Sample alignment to the zebrafish reference transcriptome ENSEMBL zv10 release 82 using “cellranger count” with default parameters to obtain a digital gene expression DGE matrix genes x cells summarizing transcript counts. Genome build: ENSEMBL zv10 release 82 Supplementary files format and content: Csv files including gene expression matrix from all larval samples and all adult samples,Zebrafish retina 4 mpf 6 mpf,All zebrafish used were anesthetized in a lethal overdose of tricaine and rapidly euthanized by immersion in ice water for 10 min. Retinas from larval or adult fish were dissected in oxygenated ox Ames and transferred into ox Ames on ice until tissue collection was completed.,Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 larval retinas or 45 minutes adult retinas. To stop the digestion the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence activated cell sorting FACS purification. Non transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within 45 minutes post FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell. 10x Chromium 3’ v2 platform 10X Genomics Pleasanton CA,Adult and larval zebrafish were maintained on a 14:10 hour light:dark cycle at 28°C. Embryos were bred in Danieau’s solution 17 mM NaCl 2 mM KCl 0.12 mM MgSO4 1.8 mM CaNO32 1.5 mM HEPES.,tissue:retina,GSM4627215,GSM4627215: Adult Zebrafish 3; Danio rerio; RNA Seq,GSM4627215,,1,Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 larval retinas or 45 minutes adult retinas. To stop the digestion the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence activated cell sorting FACS purification. Non transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within 45 minutes post FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell. 10x Chromium three prime v2 platform 10X Genomics Pleasanton CA,GEO Accession:GSM4627215,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP268010,,assembly:GRCz10|intentional duplicate,ZebrafishRGC3.bam,10X Genomics bam file,22433937540.0,228917730.0,GSM4627215 r1,0:98,A:6661186629;C:4423555909;G:4672058698;T:6668698022;N:8438282,98,,,,6661186629,4423555909,4672058698,6668698022,8438282,SRX8583205,SRS6874822,SRA1089100,GEO,"Karthik Shekhar, Chemical and Biomolecular Engineering, UC Berkeley",1,0.88295,,0.30604,,0.79228,,0.53139,,98,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-06-19,Multi-stage,Multi-stage,Eye,Sensory System 59868,SRR12054785,SRX8583204,SRS6874821,SRP268010,PRJNA640615,Molecular classification of zebrafish retinal ganglion cells links genes to behavior,GSE152842,Transcriptome Analysis,Retinal ganglion cells RGCs form an array of feature detectors which transmit visual information through segregated channels to central brain regions. Characterizing RGC diversity is required to understand the logic of this functional parcellation. Using single cell transcriptomics we systematically classified RGCs in adult and larval zebrafish thereby identifying marker genes for >30 stable and several transient cell types cell type specific markers and their maturational changes. We used this catalog to engineer transgenic driverreporter lines enabling experimental genetic access to specific RGC types. Strikingly expression of a few transcription factors often predicts dendrite morphological features in particular and axonal projections to specific tectal layers and extratectal targets. Moreover iIn vivo calcium imaging revealed that molecularly defined RGCs exhibit highly specific functional tuningphysiological properties. Finally chemogenetic ablation of eomesa+ RGCs which comprise three melanopsin expressing types with a specific projections to thalamus pretectum and one deep tectal layerpattern selectively impaired a visual behavior phototaxis. Our study establishes a framework and provides a resource for systematically studying the functional architecture of the visual system. Overall design: RGCs were labeled using transgenic Tgisl2b:tagRFP zebrafish that express RFP in all RGCs. Retinas from larval 5 dpf and adult 4 month 6 month fish were dissected and sorted using FACS. For the larval dataset 200 manually dissected retinas were dissociated in one experiment and single cell profiles were collected across 3 replicates. For the adult dataset about 20 retinas per batch were dissected and dissociated and droplet RNA sequencing was performed collecting a total of 15 replicates across 5 experiments. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell.,,pubmed:33357413,,Adult Zebrafish 2,GSM4627214,,source name:Zebrafish retina 4 mpf 6 mpf|tissue:retina,Adult Zebrafish 2,Sample demultiplexing for each of 15 adult and 3 larval samples with the mkfastq function from Cell Ranger softwareversion 2.1.0 10X Genomics Sample alignment to the zebrafish reference transcriptome ENSEMBL zv10 release 82 using “cellranger count” with default parameters to obtain a digital gene expression DGE matrix genes x cells summarizing transcript counts. Genome build: ENSEMBL zv10 release 82 Supplementary files format and content: Csv files including gene expression matrix from all larval samples and all adult samples,Zebrafish retina 4 mpf 6 mpf,All zebrafish used were anesthetized in a lethal overdose of tricaine and rapidly euthanized by immersion in ice water for 10 min. Retinas from larval or adult fish were dissected in oxygenated ox Ames and transferred into ox Ames on ice until tissue collection was completed.,Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 larval retinas or 45 minutes adult retinas. To stop the digestion the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence activated cell sorting FACS purification. Non transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within 45 minutes post FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell. 10x Chromium 3’ v2 platform 10X Genomics Pleasanton CA,Adult and larval zebrafish were maintained on a 14:10 hour light:dark cycle at 28°C. Embryos were bred in Danieau’s solution 17 mM NaCl 2 mM KCl 0.12 mM MgSO4 1.8 mM CaNO32 1.5 mM HEPES.,tissue:retina,GSM4627214,GSM4627214: Adult Zebrafish 2; Danio rerio; RNA Seq,GSM4627214,,1,Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 larval retinas or 45 minutes adult retinas. To stop the digestion the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence activated cell sorting FACS purification. Non transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within 45 minutes post FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell. 10x Chromium three prime v2 platform 10X Genomics Pleasanton CA,GEO Accession:GSM4627214,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP268010,,assembly:GRCz10|intentional duplicate,ZebrafishRGC2.bam,10X Genomics bam file,25334714412.0,258517494.0,GSM4627214 r1,0:98,A:7365387325;C:5107021128;G:5455973330;T:7396374911;N:9957718,98,,,,7365387325,5107021128,5455973330,7396374911,9957718,SRX8583204,SRS6874821,SRA1089100,GEO,"Karthik Shekhar, Chemical and Biomolecular Engineering, UC Berkeley",1,0.90152,,0.22907,,0.78776,,0.50123,,98,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-06-19,Multi-stage,Multi-stage,Eye,Sensory System 59869,SRR12054784,SRX8583203,SRS6874820,SRP268010,PRJNA640615,Molecular classification of zebrafish retinal ganglion cells links genes to behavior,GSE152842,Transcriptome Analysis,Retinal ganglion cells RGCs form an array of feature detectors which transmit visual information through segregated channels to central brain regions. Characterizing RGC diversity is required to understand the logic of this functional parcellation. Using single cell transcriptomics we systematically classified RGCs in adult and larval zebrafish thereby identifying marker genes for >30 stable and several transient cell types cell type specific markers and their maturational changes. We used this catalog to engineer transgenic driverreporter lines enabling experimental genetic access to specific RGC types. Strikingly expression of a few transcription factors often predicts dendrite morphological features in particular and axonal projections to specific tectal layers and extratectal targets. Moreover iIn vivo calcium imaging revealed that molecularly defined RGCs exhibit highly specific functional tuningphysiological properties. Finally chemogenetic ablation of eomesa+ RGCs which comprise three melanopsin expressing types with a specific projections to thalamus pretectum and one deep tectal layerpattern selectively impaired a visual behavior phototaxis. Our study establishes a framework and provides a resource for systematically studying the functional architecture of the visual system. Overall design: RGCs were labeled using transgenic Tgisl2b:tagRFP zebrafish that express RFP in all RGCs. Retinas from larval 5 dpf and adult 4 month 6 month fish were dissected and sorted using FACS. For the larval dataset 200 manually dissected retinas were dissociated in one experiment and single cell profiles were collected across 3 replicates. For the adult dataset about 20 retinas per batch were dissected and dissociated and droplet RNA sequencing was performed collecting a total of 15 replicates across 5 experiments. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell.,,pubmed:33357413,,Adult Zebrafish 1,GSM4627213,,source name:Zebrafish retina 4 mpf 6 mpf|tissue:retina,Adult Zebrafish 1,Sample demultiplexing for each of 15 adult and 3 larval samples with the mkfastq function from Cell Ranger softwareversion 2.1.0 10X Genomics Sample alignment to the zebrafish reference transcriptome ENSEMBL zv10 release 82 using “cellranger count” with default parameters to obtain a digital gene expression DGE matrix genes x cells summarizing transcript counts. Genome build: ENSEMBL zv10 release 82 Supplementary files format and content: Csv files including gene expression matrix from all larval samples and all adult samples,Zebrafish retina 4 mpf 6 mpf,All zebrafish used were anesthetized in a lethal overdose of tricaine and rapidly euthanized by immersion in ice water for 10 min. Retinas from larval or adult fish were dissected in oxygenated ox Ames and transferred into ox Ames on ice until tissue collection was completed.,Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 larval retinas or 45 minutes adult retinas. To stop the digestion the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence activated cell sorting FACS purification. Non transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within 45 minutes post FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell. 10x Chromium 3’ v2 platform 10X Genomics Pleasanton CA,Adult and larval zebrafish were maintained on a 14:10 hour light:dark cycle at 28°C. Embryos were bred in Danieau’s solution 17 mM NaCl 2 mM KCl 0.12 mM MgSO4 1.8 mM CaNO32 1.5 mM HEPES.,tissue:retina,GSM4627213,GSM4627213: Adult Zebrafish 1; Danio rerio; RNA Seq,GSM4627213,,1,Retinas were digested in papain 20U/ml in ox Ames at 28°C for 30 larval retinas or 45 minutes adult retinas. To stop the digestion the papain solution was replaced by papain inhibitor solution containing ovomucoid 15mg/ml and BSA 15mg/ml. Tissue was gently dissociated by trituration using a flamed glass pipette in papain inhibitor solution. To wash the cell suspension cells were pelleted at 250g for 8 minutes and resuspended in ox. Ames containing 0.4% BSA. The cell suspension was filtered through a 30µm strainer prior to fluorescence activated cell sorting FACS purification. Non transgenic wildtype retinas were used to determine background fluorescence levels and adjust sorting gates. Calcein blue was added to distinguish live RFP+ RGCs. Cells were washed and resuspended in PBS 0.04% BSA and loaded onto the microfluidic device within 45 minutes post FACS enrichment. Droplet RNA sequencing experiments using the 10X chromium platform were performed according to the manufacturer's instructions with no modifications. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of 30 000 reads per cell. 10x Chromium three prime v2 platform 10X Genomics Pleasanton CA,GEO Accession:GSM4627213,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,Illumina HiSeq 2500,,SRP268010,,assembly:GRCz10|intentional duplicate,ZebrafishRGC1.bam,10X Genomics bam file,25693480848.0,262178376.0,GSM4627213 r1,0:98,A:7491507845;C:5131022895;G:5429848529;T:7631356264;N:9745315,98,,,,7491507845,5131022895,5429848529,7631356264,9745315,SRX8583203,SRS6874820,SRA1089100,GEO,"Karthik Shekhar, Chemical and Biomolecular Engineering, UC Berkeley",1,0.90363,,0.22835,,0.78632,,0.48812,,98,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2020-06-19,Multi-stage,Multi-stage,Eye,Sensory System