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122 rows where experiment.library_layout = "PAIRED", technology = "bulk" and tissue_curation = "Eye"

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devstage_curation_coarse 3

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tissue_curation 1

  • Eye · 122 ✖

technology 1

  • bulk · 122 ✖

experiment.platform 1

experiment.library_strategy 1

experiment.library_source 1

experiment.library_selection 1

experiment.library_layout 1

  • PAIRED · 122 ✖
Link 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
34447 34447 SRR31768861 SRX27130600 SRS23587604 SRP552464 PRJNA1200121 Single Cell Profiling of Zebrafish Retinal Ganglion Cells Reveal an Injury Response Mediated by Cell Dedifferentiation and Leukocytes [RNA Seq] GSE284728 Transcriptome Analysis Retinal ganglion cells RGCs are the sole projection neurons connecting the retina to the brain and therefore play a critical role in vision. Death of RGCs during glaucoma results in irreversible loss of vision as RGCs do not xxx post injury in the human eye. There are no FDA approved drugs to prevent RGC death and/or promote RGC xxx post injury. There is a critical need to better understand the molecular underpinnings of neuroprotection in vivo that can then be leveraged to develop new therapeutic approaches. Unlike mammals zebrafish RGCs are resilient to injury and this study characterizes zebrafish retinal ganglion cell injury response to optic nerve transection ONT using isl2b:eGFP transgenic fish line single cell and bulk RNA sequencing and in situ hybridization. We demonstrate that zebrafish RGCs do not show subtype specific resilience to injury but show a distinct temporal injury response which includes an increase in subtype 3 a cell population having progenitor and regenerative identity. Overall design: Nine bulk RNA seq libraries were prepared from FACS purified retinal ganglion cells RGC from pooled tissue from nine sets of adult zebrafish: three libraries from injured tissue post optic nerve transection at each of 3 timepoints: 1 5 and 7 xxx post injury dpi and three from uninjured tissue on the same days.       adult zebrafish retinal ganglion cells day 7 uninjured fish set #3 GSM8691385   source name:retina|tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:uninjured|time:day 7|geo loc name:missing|collection date:missing adult zebrafish retinal ganglion cells day 7 uninjured fish set #3 Raw read data were imported to the CLC Genomics Workbench Qiagen Digital Insights licensed through the Molecular Biology Information Service of the Health Sciences Library System at the University of Pittsburgh. Differential gene expression analysis was performed on Injured versus Uninjured groups post genes with TPM=0 in one or more replicates were excluded. DEGs from 1 5 and 7 dpi time points were identified using the following filter: the maximum of the average group RPKM value >1.5 absolute fold change >1.5 FDR P value <0.05. Pathway enrichment analyses were performed using DAVID Bioinformatics Resources 6.8 https://david.ncifcrf.gov and using the KEGG database. Assembly: Danio rerio reference genome and annotations from GRCz11 from Ensembl version 106 Supplementary files format and content: Tab delimited BulkRNAseq raw counts.tsv file has raw gene counts for each Sample retina Optic nerve transection ONT was performed under dissecting microscope. The optic nerve in the left eye was severed while the right eye became the uninjured sham control. 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc. isl2b:GFP transgenic zebrafish Danio rerio used in this study were 3 6 maintained on a 14:10 hour light:dark cycle at 28C. tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:uninjured|time:day 7 GSM8691385 GSM8691385: adult zebrafish retinal ganglion cells day 7 uninjured fish set #3; Danio rerio; RNA Seq GSM8691385 r1 GSM8691385 1 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 2000   SRP552464     7DPI_uninjured_03_S30_R1_001.fastq.gz 7DPI_uninjured_03_S30_R2_001.fastq.gz fastq fastq 5698047024.0 29362783.0 GSM8691385 r1 0:97.02 1:97.03 A:1667059209;C:1124020328;G:1186053751;T:1719272359;N:1641377 97 97     1667059209 1124020328 1186053751 1719272359 1641377 SRX27130600 SRS23587604 SRA2037426 Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin                       B B biological fallback assumption illumina nextseq_v2 unknown cdna_unspecified nextera bulk bulk bulk   United States 2024-12-18 Adult Adult Eye Sensory System
34448 34448 SRR31768862 SRX27130599 SRS23587603 SRP552464 PRJNA1200121 Single Cell Profiling of Zebrafish Retinal Ganglion Cells Reveal an Injury Response Mediated by Cell Dedifferentiation and Leukocytes [RNA Seq] GSE284728 Transcriptome Analysis Retinal ganglion cells RGCs are the sole projection neurons connecting the retina to the brain and therefore play a critical role in vision. Death of RGCs during glaucoma results in irreversible loss of vision as RGCs do not xxx post injury in the human eye. There are no FDA approved drugs to prevent RGC death and/or promote RGC xxx post injury. There is a critical need to better understand the molecular underpinnings of neuroprotection in vivo that can then be leveraged to develop new therapeutic approaches. Unlike mammals zebrafish RGCs are resilient to injury and this study characterizes zebrafish retinal ganglion cell injury response to optic nerve transection ONT using isl2b:eGFP transgenic fish line single cell and bulk RNA sequencing and in situ hybridization. We demonstrate that zebrafish RGCs do not show subtype specific resilience to injury but show a distinct temporal injury response which includes an increase in subtype 3 a cell population having progenitor and regenerative identity. Overall design: Nine bulk RNA seq libraries were prepared from FACS purified retinal ganglion cells RGC from pooled tissue from nine sets of adult zebrafish: three libraries from injured tissue post optic nerve transection at each of 3 timepoints: 1 5 and 7 xxx post injury dpi and three from uninjured tissue on the same days.       adult zebrafish retinal ganglion cells day 7 uninjured fish set #2 GSM8691384   source name:retina|tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:uninjured|time:day 7|geo loc name:missing|collection date:missing adult zebrafish retinal ganglion cells day 7 uninjured fish set #2 Raw read data were imported to the CLC Genomics Workbench Qiagen Digital Insights licensed through the Molecular Biology Information Service of the Health Sciences Library System at the University of Pittsburgh. Differential gene expression analysis was performed on Injured versus Uninjured groups post genes with TPM=0 in one or more replicates were excluded. DEGs from 1 5 and 7 dpi time points were identified using the following filter: the maximum of the average group RPKM value >1.5 absolute fold change >1.5 FDR P value <0.05. Pathway enrichment analyses were performed using DAVID Bioinformatics Resources 6.8 https://david.ncifcrf.gov and using the KEGG database. Assembly: Danio rerio reference genome and annotations from GRCz11 from Ensembl version 106 Supplementary files format and content: Tab delimited BulkRNAseq raw counts.tsv file has raw gene counts for each Sample retina Optic nerve transection ONT was performed under dissecting microscope. The optic nerve in the left eye was severed while the right eye became the uninjured sham control. 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc. isl2b:GFP transgenic zebrafish Danio rerio used in this study were 3 6 maintained on a 14:10 hour light:dark cycle at 28C. tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:uninjured|time:day 7 GSM8691384 GSM8691384: adult zebrafish retinal ganglion cells day 7 uninjured fish set #2; Danio rerio; RNA Seq GSM8691384 r1 GSM8691384 1 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 2000   SRP552464     7DPI_uninjured_02_S29_R1_001.fastq.gz 7DPI_uninjured_02_S29_R2_001.fastq.gz fastq fastq 6167521979.0 31682719.0 GSM8691384 r1 0:97.33 1:97.33 A:1801911913;C:1219391300;G:1283420442;T:1861159517;N:1638807 97 97     1801911913 1219391300 1283420442 1861159517 1638807 SRX27130599 SRS23587603 SRA2037426 Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin                       B B biological fallback assumption illumina nextseq_v2 unknown cdna_unspecified nextera bulk bulk bulk   United States 2024-12-18 Adult Adult Eye Sensory System
34449 34449 SRR31768863 SRX27130598 SRS23587602 SRP552464 PRJNA1200121 Single Cell Profiling of Zebrafish Retinal Ganglion Cells Reveal an Injury Response Mediated by Cell Dedifferentiation and Leukocytes [RNA Seq] GSE284728 Transcriptome Analysis Retinal ganglion cells RGCs are the sole projection neurons connecting the retina to the brain and therefore play a critical role in vision. Death of RGCs during glaucoma results in irreversible loss of vision as RGCs do not xxx post injury in the human eye. There are no FDA approved drugs to prevent RGC death and/or promote RGC xxx post injury. There is a critical need to better understand the molecular underpinnings of neuroprotection in vivo that can then be leveraged to develop new therapeutic approaches. Unlike mammals zebrafish RGCs are resilient to injury and this study characterizes zebrafish retinal ganglion cell injury response to optic nerve transection ONT using isl2b:eGFP transgenic fish line single cell and bulk RNA sequencing and in situ hybridization. We demonstrate that zebrafish RGCs do not show subtype specific resilience to injury but show a distinct temporal injury response which includes an increase in subtype 3 a cell population having progenitor and regenerative identity. Overall design: Nine bulk RNA seq libraries were prepared from FACS purified retinal ganglion cells RGC from pooled tissue from nine sets of adult zebrafish: three libraries from injured tissue post optic nerve transection at each of 3 timepoints: 1 5 and 7 xxx post injury dpi and three from uninjured tissue on the same days.       adult zebrafish retinal ganglion cells day 7 uninjured fish set #1 GSM8691383   source name:retina|tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:uninjured|time:day 7|geo loc name:missing|collection date:missing adult zebrafish retinal ganglion cells day 7 uninjured fish set #1 Raw read data were imported to the CLC Genomics Workbench Qiagen Digital Insights licensed through the Molecular Biology Information Service of the Health Sciences Library System at the University of Pittsburgh. Differential gene expression analysis was performed on Injured versus Uninjured groups post genes with TPM=0 in one or more replicates were excluded. DEGs from 1 5 and 7 dpi time points were identified using the following filter: the maximum of the average group RPKM value >1.5 absolute fold change >1.5 FDR P value <0.05. Pathway enrichment analyses were performed using DAVID Bioinformatics Resources 6.8 https://david.ncifcrf.gov and using the KEGG database. Assembly: Danio rerio reference genome and annotations from GRCz11 from Ensembl version 106 Supplementary files format and content: Tab delimited BulkRNAseq raw counts.tsv file has raw gene counts for each Sample retina Optic nerve transection ONT was performed under dissecting microscope. The optic nerve in the left eye was severed while the right eye became the uninjured sham control. 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc. isl2b:GFP transgenic zebrafish Danio rerio used in this study were 3 6 maintained on a 14:10 hour light:dark cycle at 28C. tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:uninjured|time:day 7 GSM8691383 GSM8691383: adult zebrafish retinal ganglion cells day 7 uninjured fish set #1; Danio rerio; RNA Seq GSM8691383 r1 GSM8691383 1 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 2000   SRP552464     7DPI_uninjured_01_S28_R1_001.fastq.gz 7DPI_uninjured_01_S28_R2_001.fastq.gz fastq fastq 6017592709.0 30979971.0 GSM8691383 r1 0:97.12 1:97.12 A:1777015201;C:1171009321;G:1236169034;T:1831770286;N:1628867 97 97     1777015201 1171009321 1236169034 1831770286 1628867 SRX27130598 SRS23587602 SRA2037426 Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin                       B B biological fallback assumption illumina nextseq_v2 unknown cdna_unspecified nextera bulk bulk bulk   United States 2024-12-18 Adult Adult Eye Sensory System
34450 34450 SRR31768864 SRX27130597 SRS23587601 SRP552464 PRJNA1200121 Single Cell Profiling of Zebrafish Retinal Ganglion Cells Reveal an Injury Response Mediated by Cell Dedifferentiation and Leukocytes [RNA Seq] GSE284728 Transcriptome Analysis Retinal ganglion cells RGCs are the sole projection neurons connecting the retina to the brain and therefore play a critical role in vision. Death of RGCs during glaucoma results in irreversible loss of vision as RGCs do not xxx post injury in the human eye. There are no FDA approved drugs to prevent RGC death and/or promote RGC xxx post injury. There is a critical need to better understand the molecular underpinnings of neuroprotection in vivo that can then be leveraged to develop new therapeutic approaches. Unlike mammals zebrafish RGCs are resilient to injury and this study characterizes zebrafish retinal ganglion cell injury response to optic nerve transection ONT using isl2b:eGFP transgenic fish line single cell and bulk RNA sequencing and in situ hybridization. We demonstrate that zebrafish RGCs do not show subtype specific resilience to injury but show a distinct temporal injury response which includes an increase in subtype 3 a cell population having progenitor and regenerative identity. Overall design: Nine bulk RNA seq libraries were prepared from FACS purified retinal ganglion cells RGC from pooled tissue from nine sets of adult zebrafish: three libraries from injured tissue post optic nerve transection at each of 3 timepoints: 1 5 and 7 xxx post injury dpi and three from uninjured tissue on the same days.       adult zebrafish retinal ganglion cells 7 xxx post injury fish set #3 GSM8691382   source name:retina|tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:optic nerve transection|time:day xxx post injury|geo loc name:missing|collection date:missing adult zebrafish retinal ganglion cells 7 xxx post injury fish set #3 Raw read data were imported to the CLC Genomics Workbench Qiagen Digital Insights licensed through the Molecular Biology Information Service of the Health Sciences Library System at the University of Pittsburgh. Differential gene expression analysis was performed on Injured versus Uninjured groups post genes with TPM=0 in one or more replicates were excluded. DEGs from 1 5 and 7 dpi time points were identified using the following filter: the maximum of the average group RPKM value >1.5 absolute fold change >1.5 FDR P value <0.05. Pathway enrichment analyses were performed using DAVID Bioinformatics Resources 6.8 https://david.ncifcrf.gov and using the KEGG database. Assembly: Danio rerio reference genome and annotations from GRCz11 from Ensembl version 106 Supplementary files format and content: Tab delimited BulkRNAseq raw counts.tsv file has raw gene counts for each Sample retina Optic nerve transection ONT was performed under dissecting microscope. The optic nerve in the left eye was severed while the right eye became the uninjured sham control. 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc. isl2b:GFP transgenic zebrafish Danio rerio used in this study were 3 6 maintained on a 14:10 hour light:dark cycle at 28C. tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:optic nerve transection|time:day xxx post injury GSM8691382 GSM8691382: adult zebrafish retinal ganglion cells 7 xxx post injury fish set #3; Danio rerio; RNA Seq GSM8691382 r1 GSM8691382 1 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 2000   SRP552464     7DPI_injured_03_S27_R1_001.fastq.gz 7DPI_injured_03_S27_R2_001.fastq.gz fastq fastq 6341961565.0 32524944.0 GSM8691382 r1 0:97.49 1:97.49 A:1851283556;C:1264914896;G:1328636652;T:1895553719;N:1572742 97 97     1851283556 1264914896 1328636652 1895553719 1572742 SRX27130597 SRS23587601 SRA2037426 Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin                       B B biological fallback assumption illumina nextseq_v2 unknown cdna_unspecified nextera bulk bulk bulk   United States 2024-12-18 Adult Adult Eye Sensory System
34451 34451 SRR31768865 SRX27130596 SRS23587600 SRP552464 PRJNA1200121 Single Cell Profiling of Zebrafish Retinal Ganglion Cells Reveal an Injury Response Mediated by Cell Dedifferentiation and Leukocytes [RNA Seq] GSE284728 Transcriptome Analysis Retinal ganglion cells RGCs are the sole projection neurons connecting the retina to the brain and therefore play a critical role in vision. Death of RGCs during glaucoma results in irreversible loss of vision as RGCs do not xxx post injury in the human eye. There are no FDA approved drugs to prevent RGC death and/or promote RGC xxx post injury. There is a critical need to better understand the molecular underpinnings of neuroprotection in vivo that can then be leveraged to develop new therapeutic approaches. Unlike mammals zebrafish RGCs are resilient to injury and this study characterizes zebrafish retinal ganglion cell injury response to optic nerve transection ONT using isl2b:eGFP transgenic fish line single cell and bulk RNA sequencing and in situ hybridization. We demonstrate that zebrafish RGCs do not show subtype specific resilience to injury but show a distinct temporal injury response which includes an increase in subtype 3 a cell population having progenitor and regenerative identity. Overall design: Nine bulk RNA seq libraries were prepared from FACS purified retinal ganglion cells RGC from pooled tissue from nine sets of adult zebrafish: three libraries from injured tissue post optic nerve transection at each of 3 timepoints: 1 5 and 7 xxx post injury dpi and three from uninjured tissue on the same days.       adult zebrafish retinal ganglion cells 7 xxx post injury fish set #2 GSM8691381   source name:retina|tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:optic nerve transection|time:day xxx post injury|geo loc name:missing|collection date:missing adult zebrafish retinal ganglion cells 7 xxx post injury fish set #2 Raw read data were imported to the CLC Genomics Workbench Qiagen Digital Insights licensed through the Molecular Biology Information Service of the Health Sciences Library System at the University of Pittsburgh. Differential gene expression analysis was performed on Injured versus Uninjured groups post genes with TPM=0 in one or more replicates were excluded. DEGs from 1 5 and 7 dpi time points were identified using the following filter: the maximum of the average group RPKM value >1.5 absolute fold change >1.5 FDR P value <0.05. Pathway enrichment analyses were performed using DAVID Bioinformatics Resources 6.8 https://david.ncifcrf.gov and using the KEGG database. Assembly: Danio rerio reference genome and annotations from GRCz11 from Ensembl version 106 Supplementary files format and content: Tab delimited BulkRNAseq raw counts.tsv file has raw gene counts for each Sample retina Optic nerve transection ONT was performed under dissecting microscope. The optic nerve in the left eye was severed while the right eye became the uninjured sham control. 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc. isl2b:GFP transgenic zebrafish Danio rerio used in this study were 3 6 maintained on a 14:10 hour light:dark cycle at 28C. tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:optic nerve transection|time:day xxx post injury GSM8691381 GSM8691381: adult zebrafish retinal ganglion cells 7 xxx post injury fish set #2; Danio rerio; RNA Seq GSM8691381 r1 GSM8691381 1 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 2000   SRP552464     7DPI_injured_02_S26_R1_001.fastq.gz 7DPI_injured_02_S26_R2_001.fastq.gz fastq fastq 6235870535.0 31994279.0 GSM8691381 r1 0:97.45 1:97.46 A:1822480684;C:1235794789;G:1300588308;T:1875414630;N:1592124 97 97     1822480684 1235794789 1300588308 1875414630 1592124 SRX27130596 SRS23587600 SRA2037426 Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin                       B B biological fallback assumption illumina nextseq_v2 unknown cdna_unspecified nextera bulk bulk bulk   United States 2024-12-18 Adult Adult Eye Sensory System
34452 34452 SRR31768866 SRX27130595 SRS23587599 SRP552464 PRJNA1200121 Single Cell Profiling of Zebrafish Retinal Ganglion Cells Reveal an Injury Response Mediated by Cell Dedifferentiation and Leukocytes [RNA Seq] GSE284728 Transcriptome Analysis Retinal ganglion cells RGCs are the sole projection neurons connecting the retina to the brain and therefore play a critical role in vision. Death of RGCs during glaucoma results in irreversible loss of vision as RGCs do not xxx post injury in the human eye. There are no FDA approved drugs to prevent RGC death and/or promote RGC xxx post injury. There is a critical need to better understand the molecular underpinnings of neuroprotection in vivo that can then be leveraged to develop new therapeutic approaches. Unlike mammals zebrafish RGCs are resilient to injury and this study characterizes zebrafish retinal ganglion cell injury response to optic nerve transection ONT using isl2b:eGFP transgenic fish line single cell and bulk RNA sequencing and in situ hybridization. We demonstrate that zebrafish RGCs do not show subtype specific resilience to injury but show a distinct temporal injury response which includes an increase in subtype 3 a cell population having progenitor and regenerative identity. Overall design: Nine bulk RNA seq libraries were prepared from FACS purified retinal ganglion cells RGC from pooled tissue from nine sets of adult zebrafish: three libraries from injured tissue post optic nerve transection at each of 3 timepoints: 1 5 and 7 xxx post injury dpi and three from uninjured tissue on the same days.       adult zebrafish retinal ganglion cells 7 xxx post injury fish set #1 GSM8691380   source name:retina|tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:optic nerve transection|time:day xxx post injury|geo loc name:missing|collection date:missing adult zebrafish retinal ganglion cells 7 xxx post injury fish set #1 Raw read data were imported to the CLC Genomics Workbench Qiagen Digital Insights licensed through the Molecular Biology Information Service of the Health Sciences Library System at the University of Pittsburgh. Differential gene expression analysis was performed on Injured versus Uninjured groups post genes with TPM=0 in one or more replicates were excluded. DEGs from 1 5 and 7 dpi time points were identified using the following filter: the maximum of the average group RPKM value >1.5 absolute fold change >1.5 FDR P value <0.05. Pathway enrichment analyses were performed using DAVID Bioinformatics Resources 6.8 https://david.ncifcrf.gov and using the KEGG database. Assembly: Danio rerio reference genome and annotations from GRCz11 from Ensembl version 106 Supplementary files format and content: Tab delimited BulkRNAseq raw counts.tsv file has raw gene counts for each Sample retina Optic nerve transection ONT was performed under dissecting microscope. The optic nerve in the left eye was severed while the right eye became the uninjured sham control. 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc. isl2b:GFP transgenic zebrafish Danio rerio used in this study were 3 6 maintained on a 14:10 hour light:dark cycle at 28C. tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:optic nerve transection|time:day xxx post injury GSM8691380 GSM8691380: adult zebrafish retinal ganglion cells 7 xxx post injury fish set #1; Danio rerio; RNA Seq GSM8691380 r1 GSM8691380 1 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 2000   SRP552464     7DPI_injured_01_S25_R1_001.fastq.gz 7DPI_injured_01_S25_R2_001.fastq.gz fastq fastq 6174385200.0 31622915.0 GSM8691380 r1 0:97.62 1:97.63 A:1787576071;C:1247968531;G:1306271111;T:1831148003;N:1421484 97 97     1787576071 1247968531 1306271111 1831148003 1421484 SRX27130595 SRS23587599 SRA2037426 Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin                       B B biological fallback assumption illumina nextseq_v2 unknown cdna_unspecified nextera bulk bulk bulk   United States 2024-12-18 Adult Adult Eye Sensory System
34453 34453 SRR31768867 SRX27130594 SRS23587598 SRP552464 PRJNA1200121 Single Cell Profiling of Zebrafish Retinal Ganglion Cells Reveal an Injury Response Mediated by Cell Dedifferentiation and Leukocytes [RNA Seq] GSE284728 Transcriptome Analysis Retinal ganglion cells RGCs are the sole projection neurons connecting the retina to the brain and therefore play a critical role in vision. Death of RGCs during glaucoma results in irreversible loss of vision as RGCs do not xxx post injury in the human eye. There are no FDA approved drugs to prevent RGC death and/or promote RGC xxx post injury. There is a critical need to better understand the molecular underpinnings of neuroprotection in vivo that can then be leveraged to develop new therapeutic approaches. Unlike mammals zebrafish RGCs are resilient to injury and this study characterizes zebrafish retinal ganglion cell injury response to optic nerve transection ONT using isl2b:eGFP transgenic fish line single cell and bulk RNA sequencing and in situ hybridization. We demonstrate that zebrafish RGCs do not show subtype specific resilience to injury but show a distinct temporal injury response which includes an increase in subtype 3 a cell population having progenitor and regenerative identity. Overall design: Nine bulk RNA seq libraries were prepared from FACS purified retinal ganglion cells RGC from pooled tissue from nine sets of adult zebrafish: three libraries from injured tissue post optic nerve transection at each of 3 timepoints: 1 5 and 7 xxx post injury dpi and three from uninjured tissue on the same days.       adult zebrafish retinal ganglion cells day 5 uninjured fish set #3 GSM8691379   source name:retina|tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:uninjured|time:day 5|geo loc name:missing|collection date:missing adult zebrafish retinal ganglion cells day 5 uninjured fish set #3 Raw read data were imported to the CLC Genomics Workbench Qiagen Digital Insights licensed through the Molecular Biology Information Service of the Health Sciences Library System at the University of Pittsburgh. Differential gene expression analysis was performed on Injured versus Uninjured groups post genes with TPM=0 in one or more replicates were excluded. DEGs from 1 5 and 7 dpi time points were identified using the following filter: the maximum of the average group RPKM value >1.5 absolute fold change >1.5 FDR P value <0.05. Pathway enrichment analyses were performed using DAVID Bioinformatics Resources 6.8 https://david.ncifcrf.gov and using the KEGG database. Assembly: Danio rerio reference genome and annotations from GRCz11 from Ensembl version 106 Supplementary files format and content: Tab delimited BulkRNAseq raw counts.tsv file has raw gene counts for each Sample retina Optic nerve transection ONT was performed under dissecting microscope. The optic nerve in the left eye was severed while the right eye became the uninjured sham control. 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc. isl2b:GFP transgenic zebrafish Danio rerio used in this study were 3 6 maintained on a 14:10 hour light:dark cycle at 28C. tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:uninjured|time:day 5 GSM8691379 GSM8691379: adult zebrafish retinal ganglion cells day 5 uninjured fish set #3; Danio rerio; RNA Seq GSM8691379 r1 GSM8691379 1 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 2000   SRP552464     5DPI_uninjured_03_S24_R1_001.fastq.gz 5DPI_uninjured_03_S24_R2_001.fastq.gz fastq fastq 6412343629.0 34154945.0 GSM8691379 r1 0:93.86 1:93.88 A:1885512926;C:1263616925;G:1311969388;T:1947630859;N:3613531 93 93     1885512926 1263616925 1311969388 1947630859 3613531 SRX27130594 SRS23587598 SRA2037426 Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin                       B B biological fallback assumption illumina nextseq_v2 unknown cdna_unspecified nextera bulk bulk bulk   United States 2024-12-18 Adult Adult Eye Sensory System
34454 34454 SRR31768868 SRX27130593 SRS23587597 SRP552464 PRJNA1200121 Single Cell Profiling of Zebrafish Retinal Ganglion Cells Reveal an Injury Response Mediated by Cell Dedifferentiation and Leukocytes [RNA Seq] GSE284728 Transcriptome Analysis Retinal ganglion cells RGCs are the sole projection neurons connecting the retina to the brain and therefore play a critical role in vision. Death of RGCs during glaucoma results in irreversible loss of vision as RGCs do not xxx post injury in the human eye. There are no FDA approved drugs to prevent RGC death and/or promote RGC xxx post injury. There is a critical need to better understand the molecular underpinnings of neuroprotection in vivo that can then be leveraged to develop new therapeutic approaches. Unlike mammals zebrafish RGCs are resilient to injury and this study characterizes zebrafish retinal ganglion cell injury response to optic nerve transection ONT using isl2b:eGFP transgenic fish line single cell and bulk RNA sequencing and in situ hybridization. We demonstrate that zebrafish RGCs do not show subtype specific resilience to injury but show a distinct temporal injury response which includes an increase in subtype 3 a cell population having progenitor and regenerative identity. Overall design: Nine bulk RNA seq libraries were prepared from FACS purified retinal ganglion cells RGC from pooled tissue from nine sets of adult zebrafish: three libraries from injured tissue post optic nerve transection at each of 3 timepoints: 1 5 and 7 xxx post injury dpi and three from uninjured tissue on the same days.       adult zebrafish retinal ganglion cells day 5 uninjured fish set #2 GSM8691378   source name:retina|tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:uninjured|time:day 5|geo loc name:missing|collection date:missing adult zebrafish retinal ganglion cells day 5 uninjured fish set #2 Raw read data were imported to the CLC Genomics Workbench Qiagen Digital Insights licensed through the Molecular Biology Information Service of the Health Sciences Library System at the University of Pittsburgh. Differential gene expression analysis was performed on Injured versus Uninjured groups post genes with TPM=0 in one or more replicates were excluded. DEGs from 1 5 and 7 dpi time points were identified using the following filter: the maximum of the average group RPKM value >1.5 absolute fold change >1.5 FDR P value <0.05. Pathway enrichment analyses were performed using DAVID Bioinformatics Resources 6.8 https://david.ncifcrf.gov and using the KEGG database. Assembly: Danio rerio reference genome and annotations from GRCz11 from Ensembl version 106 Supplementary files format and content: Tab delimited BulkRNAseq raw counts.tsv file has raw gene counts for each Sample retina Optic nerve transection ONT was performed under dissecting microscope. The optic nerve in the left eye was severed while the right eye became the uninjured sham control. 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc. isl2b:GFP transgenic zebrafish Danio rerio used in this study were 3 6 maintained on a 14:10 hour light:dark cycle at 28C. tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:uninjured|time:day 5 GSM8691378 GSM8691378: adult zebrafish retinal ganglion cells day 5 uninjured fish set #2; Danio rerio; RNA Seq GSM8691378 r1 GSM8691378 1 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 2000   SRP552464     5DPI_uninjured_02_S23_R1_001.fastq.gz 5DPI_uninjured_02_S23_R2_001.fastq.gz fastq fastq 6672888700.0 34007957.0 GSM8691378 r1 0:98.11 1:98.11 A:1934192871;C:1346688691;G:1405041934;T:1985769861;N:1195343 98 98     1934192871 1346688691 1405041934 1985769861 1195343 SRX27130593 SRS23587597 SRA2037426 Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin                       B B biological fallback assumption illumina nextseq_v2 unknown cdna_unspecified nextera bulk bulk bulk   United States 2024-12-18 Adult Adult Eye Sensory System
34455 34455 SRR31768869 SRX27130592 SRS23587596 SRP552464 PRJNA1200121 Single Cell Profiling of Zebrafish Retinal Ganglion Cells Reveal an Injury Response Mediated by Cell Dedifferentiation and Leukocytes [RNA Seq] GSE284728 Transcriptome Analysis Retinal ganglion cells RGCs are the sole projection neurons connecting the retina to the brain and therefore play a critical role in vision. Death of RGCs during glaucoma results in irreversible loss of vision as RGCs do not xxx post injury in the human eye. There are no FDA approved drugs to prevent RGC death and/or promote RGC xxx post injury. There is a critical need to better understand the molecular underpinnings of neuroprotection in vivo that can then be leveraged to develop new therapeutic approaches. Unlike mammals zebrafish RGCs are resilient to injury and this study characterizes zebrafish retinal ganglion cell injury response to optic nerve transection ONT using isl2b:eGFP transgenic fish line single cell and bulk RNA sequencing and in situ hybridization. We demonstrate that zebrafish RGCs do not show subtype specific resilience to injury but show a distinct temporal injury response which includes an increase in subtype 3 a cell population having progenitor and regenerative identity. Overall design: Nine bulk RNA seq libraries were prepared from FACS purified retinal ganglion cells RGC from pooled tissue from nine sets of adult zebrafish: three libraries from injured tissue post optic nerve transection at each of 3 timepoints: 1 5 and 7 xxx post injury dpi and three from uninjured tissue on the same days.       adult zebrafish retinal ganglion cells day 5 uninjured fish set #1 GSM8691377   source name:retina|tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:uninjured|time:day 5|geo loc name:missing|collection date:missing adult zebrafish retinal ganglion cells day 5 uninjured fish set #1 Raw read data were imported to the CLC Genomics Workbench Qiagen Digital Insights licensed through the Molecular Biology Information Service of the Health Sciences Library System at the University of Pittsburgh. Differential gene expression analysis was performed on Injured versus Uninjured groups post genes with TPM=0 in one or more replicates were excluded. DEGs from 1 5 and 7 dpi time points were identified using the following filter: the maximum of the average group RPKM value >1.5 absolute fold change >1.5 FDR P value <0.05. Pathway enrichment analyses were performed using DAVID Bioinformatics Resources 6.8 https://david.ncifcrf.gov and using the KEGG database. Assembly: Danio rerio reference genome and annotations from GRCz11 from Ensembl version 106 Supplementary files format and content: Tab delimited BulkRNAseq raw counts.tsv file has raw gene counts for each Sample retina Optic nerve transection ONT was performed under dissecting microscope. The optic nerve in the left eye was severed while the right eye became the uninjured sham control. 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc. isl2b:GFP transgenic zebrafish Danio rerio used in this study were 3 6 maintained on a 14:10 hour light:dark cycle at 28C. tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:uninjured|time:day 5 GSM8691377 GSM8691377: adult zebrafish retinal ganglion cells day 5 uninjured fish set #1; Danio rerio; RNA Seq GSM8691377 r1 GSM8691377 1 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 2000   SRP552464     5DPI_uninjured_01_S22_R1_001.fastq.gz 5DPI_uninjured_01_S22_R2_001.fastq.gz fastq fastq 6710121204.0 35640240.0 GSM8691377 r1 0:94.13 1:94.14 A:1930817182;C:1378539334;G:1418299809;T:1979047683;N:3417196 94 94     1930817182 1378539334 1418299809 1979047683 3417196 SRX27130592 SRS23587596 SRA2037426 Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin                       B B biological fallback assumption illumina nextseq_v2 unknown cdna_unspecified nextera bulk bulk bulk   United States 2024-12-18 Adult Adult Eye Sensory System
34456 34456 SRR31768870 SRX27130591 SRS23587595 SRP552464 PRJNA1200121 Single Cell Profiling of Zebrafish Retinal Ganglion Cells Reveal an Injury Response Mediated by Cell Dedifferentiation and Leukocytes [RNA Seq] GSE284728 Transcriptome Analysis Retinal ganglion cells RGCs are the sole projection neurons connecting the retina to the brain and therefore play a critical role in vision. Death of RGCs during glaucoma results in irreversible loss of vision as RGCs do not xxx post injury in the human eye. There are no FDA approved drugs to prevent RGC death and/or promote RGC xxx post injury. There is a critical need to better understand the molecular underpinnings of neuroprotection in vivo that can then be leveraged to develop new therapeutic approaches. Unlike mammals zebrafish RGCs are resilient to injury and this study characterizes zebrafish retinal ganglion cell injury response to optic nerve transection ONT using isl2b:eGFP transgenic fish line single cell and bulk RNA sequencing and in situ hybridization. We demonstrate that zebrafish RGCs do not show subtype specific resilience to injury but show a distinct temporal injury response which includes an increase in subtype 3 a cell population having progenitor and regenerative identity. Overall design: Nine bulk RNA seq libraries were prepared from FACS purified retinal ganglion cells RGC from pooled tissue from nine sets of adult zebrafish: three libraries from injured tissue post optic nerve transection at each of 3 timepoints: 1 5 and 7 xxx post injury dpi and three from uninjured tissue on the same days.       adult zebrafish retinal ganglion cells 5 xxx post injury fish set #3 GSM8691376   source name:retina|tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:optic nerve transection|time:day xxx post injury|geo loc name:missing|collection date:missing adult zebrafish retinal ganglion cells 5 xxx post injury fish set #3 Raw read data were imported to the CLC Genomics Workbench Qiagen Digital Insights licensed through the Molecular Biology Information Service of the Health Sciences Library System at the University of Pittsburgh. Differential gene expression analysis was performed on Injured versus Uninjured groups post genes with TPM=0 in one or more replicates were excluded. DEGs from 1 5 and 7 dpi time points were identified using the following filter: the maximum of the average group RPKM value >1.5 absolute fold change >1.5 FDR P value <0.05. Pathway enrichment analyses were performed using DAVID Bioinformatics Resources 6.8 https://david.ncifcrf.gov and using the KEGG database. Assembly: Danio rerio reference genome and annotations from GRCz11 from Ensembl version 106 Supplementary files format and content: Tab delimited BulkRNAseq raw counts.tsv file has raw gene counts for each Sample retina Optic nerve transection ONT was performed under dissecting microscope. The optic nerve in the left eye was severed while the right eye became the uninjured sham control. 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc. isl2b:GFP transgenic zebrafish Danio rerio used in this study were 3 6 maintained on a 14:10 hour light:dark cycle at 28C. tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:optic nerve transection|time:day xxx post injury GSM8691376 GSM8691376: adult zebrafish retinal ganglion cells 5 xxx post injury fish set #3; Danio rerio; RNA Seq GSM8691376 r1 GSM8691376 1 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 2000   SRP552464     5DPI_injured_03_S21_R1_001.fastq.gz 5DPI_injured_03_S21_R2_001.fastq.gz fastq fastq 5815478166.0 29829330.0 GSM8691376 r1 0:97.47 1:97.49 A:1686233316;C:1173233216;G:1223299576;T:1731320900;N:1391158 97 97     1686233316 1173233216 1223299576 1731320900 1391158 SRX27130591 SRS23587595 SRA2037426 Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin                       B B biological fallback assumption illumina nextseq_v2 unknown cdna_unspecified nextera bulk bulk bulk   United States 2024-12-18 Adult Adult Eye Sensory System
34457 34457 SRR31768871 SRX27130590 SRS23587594 SRP552464 PRJNA1200121 Single Cell Profiling of Zebrafish Retinal Ganglion Cells Reveal an Injury Response Mediated by Cell Dedifferentiation and Leukocytes [RNA Seq] GSE284728 Transcriptome Analysis Retinal ganglion cells RGCs are the sole projection neurons connecting the retina to the brain and therefore play a critical role in vision. Death of RGCs during glaucoma results in irreversible loss of vision as RGCs do not xxx post injury in the human eye. There are no FDA approved drugs to prevent RGC death and/or promote RGC xxx post injury. There is a critical need to better understand the molecular underpinnings of neuroprotection in vivo that can then be leveraged to develop new therapeutic approaches. Unlike mammals zebrafish RGCs are resilient to injury and this study characterizes zebrafish retinal ganglion cell injury response to optic nerve transection ONT using isl2b:eGFP transgenic fish line single cell and bulk RNA sequencing and in situ hybridization. We demonstrate that zebrafish RGCs do not show subtype specific resilience to injury but show a distinct temporal injury response which includes an increase in subtype 3 a cell population having progenitor and regenerative identity. Overall design: Nine bulk RNA seq libraries were prepared from FACS purified retinal ganglion cells RGC from pooled tissue from nine sets of adult zebrafish: three libraries from injured tissue post optic nerve transection at each of 3 timepoints: 1 5 and 7 xxx post injury dpi and three from uninjured tissue on the same days.       adult zebrafish retinal ganglion cells 5 xxx post injury fish set #2 GSM8691375   source name:retina|tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:optic nerve transection|time:day xxx post injury|geo loc name:missing|collection date:missing adult zebrafish retinal ganglion cells 5 xxx post injury fish set #2 Raw read data were imported to the CLC Genomics Workbench Qiagen Digital Insights licensed through the Molecular Biology Information Service of the Health Sciences Library System at the University of Pittsburgh. Differential gene expression analysis was performed on Injured versus Uninjured groups post genes with TPM=0 in one or more replicates were excluded. DEGs from 1 5 and 7 dpi time points were identified using the following filter: the maximum of the average group RPKM value >1.5 absolute fold change >1.5 FDR P value <0.05. Pathway enrichment analyses were performed using DAVID Bioinformatics Resources 6.8 https://david.ncifcrf.gov and using the KEGG database. Assembly: Danio rerio reference genome and annotations from GRCz11 from Ensembl version 106 Supplementary files format and content: Tab delimited BulkRNAseq raw counts.tsv file has raw gene counts for each Sample retina Optic nerve transection ONT was performed under dissecting microscope. The optic nerve in the left eye was severed while the right eye became the uninjured sham control. 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc. isl2b:GFP transgenic zebrafish Danio rerio used in this study were 3 6 maintained on a 14:10 hour light:dark cycle at 28C. tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:optic nerve transection|time:day xxx post injury GSM8691375 GSM8691375: adult zebrafish retinal ganglion cells 5 xxx post injury fish set #2; Danio rerio; RNA Seq GSM8691375 r1 GSM8691375 1 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 2000   SRP552464     5DPI_injured_02_S20_R1_001.fastq.gz 5DPI_injured_02_S20_R2_001.fastq.gz fastq fastq 6463184463.0 33031735.0 GSM8691375 r1 0:97.83 1:97.84 A:1869116597;C:1303631450;G:1365056296;T:1924007964;N:1372156 97 97     1869116597 1303631450 1365056296 1924007964 1372156 SRX27130590 SRS23587594 SRA2037426 Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin                       B B biological fallback assumption illumina nextseq_v2 unknown cdna_unspecified nextera bulk bulk bulk   United States 2024-12-18 Adult Adult Eye Sensory System
34458 34458 SRR31768872 SRX27130589 SRS23587593 SRP552464 PRJNA1200121 Single Cell Profiling of Zebrafish Retinal Ganglion Cells Reveal an Injury Response Mediated by Cell Dedifferentiation and Leukocytes [RNA Seq] GSE284728 Transcriptome Analysis Retinal ganglion cells RGCs are the sole projection neurons connecting the retina to the brain and therefore play a critical role in vision. Death of RGCs during glaucoma results in irreversible loss of vision as RGCs do not xxx post injury in the human eye. There are no FDA approved drugs to prevent RGC death and/or promote RGC xxx post injury. There is a critical need to better understand the molecular underpinnings of neuroprotection in vivo that can then be leveraged to develop new therapeutic approaches. Unlike mammals zebrafish RGCs are resilient to injury and this study characterizes zebrafish retinal ganglion cell injury response to optic nerve transection ONT using isl2b:eGFP transgenic fish line single cell and bulk RNA sequencing and in situ hybridization. We demonstrate that zebrafish RGCs do not show subtype specific resilience to injury but show a distinct temporal injury response which includes an increase in subtype 3 a cell population having progenitor and regenerative identity. Overall design: Nine bulk RNA seq libraries were prepared from FACS purified retinal ganglion cells RGC from pooled tissue from nine sets of adult zebrafish: three libraries from injured tissue post optic nerve transection at each of 3 timepoints: 1 5 and 7 xxx post injury dpi and three from uninjured tissue on the same days.       adult zebrafish retinal ganglion cells 5 xxx post injury fish set #1 GSM8691374   source name:retina|tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:optic nerve transection|time:day xxx post injury|geo loc name:missing|collection date:missing adult zebrafish retinal ganglion cells 5 xxx post injury fish set #1 Raw read data were imported to the CLC Genomics Workbench Qiagen Digital Insights licensed through the Molecular Biology Information Service of the Health Sciences Library System at the University of Pittsburgh. Differential gene expression analysis was performed on Injured versus Uninjured groups post genes with TPM=0 in one or more replicates were excluded. DEGs from 1 5 and 7 dpi time points were identified using the following filter: the maximum of the average group RPKM value >1.5 absolute fold change >1.5 FDR P value <0.05. Pathway enrichment analyses were performed using DAVID Bioinformatics Resources 6.8 https://david.ncifcrf.gov and using the KEGG database. Assembly: Danio rerio reference genome and annotations from GRCz11 from Ensembl version 106 Supplementary files format and content: Tab delimited BulkRNAseq raw counts.tsv file has raw gene counts for each Sample retina Optic nerve transection ONT was performed under dissecting microscope. The optic nerve in the left eye was severed while the right eye became the uninjured sham control. 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc. isl2b:GFP transgenic zebrafish Danio rerio used in this study were 3 6 maintained on a 14:10 hour light:dark cycle at 28C. tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:optic nerve transection|time:day xxx post injury GSM8691374 GSM8691374: adult zebrafish retinal ganglion cells 5 xxx post injury fish set #1; Danio rerio; RNA Seq GSM8691374 r1 GSM8691374 1 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 2000   SRP552464     5DPI_injured_01_S19_R1_001.fastq.gz 5DPI_injured_01_S19_R2_001.fastq.gz fastq fastq 5770277062.0 29421167.0 GSM8691374 r1 0:98.06 1:98.07 A:1659818839;C:1176285041;G:1230578027;T:1702467577;N:1127578 98 98     1659818839 1176285041 1230578027 1702467577 1127578 SRX27130589 SRS23587593 SRA2037426 Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin                       B B biological fallback assumption illumina nextseq_v2 unknown cdna_unspecified nextera bulk bulk bulk   United States 2024-12-18 Adult Adult Eye Sensory System
34459 34459 SRR31768873 SRX27130588 SRS23587592 SRP552464 PRJNA1200121 Single Cell Profiling of Zebrafish Retinal Ganglion Cells Reveal an Injury Response Mediated by Cell Dedifferentiation and Leukocytes [RNA Seq] GSE284728 Transcriptome Analysis Retinal ganglion cells RGCs are the sole projection neurons connecting the retina to the brain and therefore play a critical role in vision. Death of RGCs during glaucoma results in irreversible loss of vision as RGCs do not xxx post injury in the human eye. There are no FDA approved drugs to prevent RGC death and/or promote RGC xxx post injury. There is a critical need to better understand the molecular underpinnings of neuroprotection in vivo that can then be leveraged to develop new therapeutic approaches. Unlike mammals zebrafish RGCs are resilient to injury and this study characterizes zebrafish retinal ganglion cell injury response to optic nerve transection ONT using isl2b:eGFP transgenic fish line single cell and bulk RNA sequencing and in situ hybridization. We demonstrate that zebrafish RGCs do not show subtype specific resilience to injury but show a distinct temporal injury response which includes an increase in subtype 3 a cell population having progenitor and regenerative identity. Overall design: Nine bulk RNA seq libraries were prepared from FACS purified retinal ganglion cells RGC from pooled tissue from nine sets of adult zebrafish: three libraries from injured tissue post optic nerve transection at each of 3 timepoints: 1 5 and 7 xxx post injury dpi and three from uninjured tissue on the same days.       adult zebrafish retinal ganglion cells day 1 uninjured fish set #3 GSM8691373   source name:retina|tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:uninjured|time:day 1|geo loc name:missing|collection date:missing adult zebrafish retinal ganglion cells day 1 uninjured fish set #3 Raw read data were imported to the CLC Genomics Workbench Qiagen Digital Insights licensed through the Molecular Biology Information Service of the Health Sciences Library System at the University of Pittsburgh. Differential gene expression analysis was performed on Injured versus Uninjured groups post genes with TPM=0 in one or more replicates were excluded. DEGs from 1 5 and 7 dpi time points were identified using the following filter: the maximum of the average group RPKM value >1.5 absolute fold change >1.5 FDR P value <0.05. Pathway enrichment analyses were performed using DAVID Bioinformatics Resources 6.8 https://david.ncifcrf.gov and using the KEGG database. Assembly: Danio rerio reference genome and annotations from GRCz11 from Ensembl version 106 Supplementary files format and content: Tab delimited BulkRNAseq raw counts.tsv file has raw gene counts for each Sample retina Optic nerve transection ONT was performed under dissecting microscope. The optic nerve in the left eye was severed while the right eye became the uninjured sham control. 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc. isl2b:GFP transgenic zebrafish Danio rerio used in this study were 3 6 maintained on a 14:10 hour light:dark cycle at 28C. tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:uninjured|time:day 1 GSM8691373 GSM8691373: adult zebrafish retinal ganglion cells day 1 uninjured fish set #3; Danio rerio; RNA Seq GSM8691373 r1 GSM8691373 1 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 2000   SRP552464     1DPI_uninjured_03_S18_R1_001.fastq.gz 1DPI_uninjured_03_S18_R2_001.fastq.gz fastq fastq 6268800701.0 32414990.0 GSM8691373 r1 0:96.68 1:96.71 A:1808208631;C:1259950394;G:1326103117;T:1872600468;N:1938091 96 96     1808208631 1259950394 1326103117 1872600468 1938091 SRX27130588 SRS23587592 SRA2037426 Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin                       B B biological fallback assumption illumina nextseq_v2 unknown cdna_unspecified nextera bulk bulk bulk   United States 2024-12-18 Adult Adult Eye Sensory System
34460 34460 SRR31768874 SRX27130587 SRS23587591 SRP552464 PRJNA1200121 Single Cell Profiling of Zebrafish Retinal Ganglion Cells Reveal an Injury Response Mediated by Cell Dedifferentiation and Leukocytes [RNA Seq] GSE284728 Transcriptome Analysis Retinal ganglion cells RGCs are the sole projection neurons connecting the retina to the brain and therefore play a critical role in vision. Death of RGCs during glaucoma results in irreversible loss of vision as RGCs do not xxx post injury in the human eye. There are no FDA approved drugs to prevent RGC death and/or promote RGC xxx post injury. There is a critical need to better understand the molecular underpinnings of neuroprotection in vivo that can then be leveraged to develop new therapeutic approaches. Unlike mammals zebrafish RGCs are resilient to injury and this study characterizes zebrafish retinal ganglion cell injury response to optic nerve transection ONT using isl2b:eGFP transgenic fish line single cell and bulk RNA sequencing and in situ hybridization. We demonstrate that zebrafish RGCs do not show subtype specific resilience to injury but show a distinct temporal injury response which includes an increase in subtype 3 a cell population having progenitor and regenerative identity. Overall design: Nine bulk RNA seq libraries were prepared from FACS purified retinal ganglion cells RGC from pooled tissue from nine sets of adult zebrafish: three libraries from injured tissue post optic nerve transection at each of 3 timepoints: 1 5 and 7 xxx post injury dpi and three from uninjured tissue on the same days.       adult zebrafish retinal ganglion cells day 1 uninjured fish set #2 GSM8691372   source name:retina|tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:uninjured|time:day 1|geo loc name:missing|collection date:missing adult zebrafish retinal ganglion cells day 1 uninjured fish set #2 Raw read data were imported to the CLC Genomics Workbench Qiagen Digital Insights licensed through the Molecular Biology Information Service of the Health Sciences Library System at the University of Pittsburgh. Differential gene expression analysis was performed on Injured versus Uninjured groups post genes with TPM=0 in one or more replicates were excluded. DEGs from 1 5 and 7 dpi time points were identified using the following filter: the maximum of the average group RPKM value >1.5 absolute fold change >1.5 FDR P value <0.05. Pathway enrichment analyses were performed using DAVID Bioinformatics Resources 6.8 https://david.ncifcrf.gov and using the KEGG database. Assembly: Danio rerio reference genome and annotations from GRCz11 from Ensembl version 106 Supplementary files format and content: Tab delimited BulkRNAseq raw counts.tsv file has raw gene counts for each Sample retina Optic nerve transection ONT was performed under dissecting microscope. The optic nerve in the left eye was severed while the right eye became the uninjured sham control. 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc. isl2b:GFP transgenic zebrafish Danio rerio used in this study were 3 6 maintained on a 14:10 hour light:dark cycle at 28C. tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:uninjured|time:day 1 GSM8691372 GSM8691372: adult zebrafish retinal ganglion cells day 1 uninjured fish set #2; Danio rerio; RNA Seq GSM8691372 r1 GSM8691372 1 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 2000   SRP552464     1DPI_uninjured_02_S17_R1_001.fastq.gz 1DPI_uninjured_02_S17_R2_001.fastq.gz fastq fastq 6475037338.0 33641146.0 GSM8691372 r1 0:96.23 1:96.24 A:1875658518;C:1291113851;G:1361172356;T:1944790051;N:2302562 96 96     1875658518 1291113851 1361172356 1944790051 2302562 SRX27130587 SRS23587591 SRA2037426 Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin                       B B biological fallback assumption illumina nextseq_v2 unknown cdna_unspecified nextera bulk bulk bulk   United States 2024-12-18 Adult Adult Eye Sensory System
34461 34461 SRR31768875 SRX27130586 SRS23587590 SRP552464 PRJNA1200121 Single Cell Profiling of Zebrafish Retinal Ganglion Cells Reveal an Injury Response Mediated by Cell Dedifferentiation and Leukocytes [RNA Seq] GSE284728 Transcriptome Analysis Retinal ganglion cells RGCs are the sole projection neurons connecting the retina to the brain and therefore play a critical role in vision. Death of RGCs during glaucoma results in irreversible loss of vision as RGCs do not xxx post injury in the human eye. There are no FDA approved drugs to prevent RGC death and/or promote RGC xxx post injury. There is a critical need to better understand the molecular underpinnings of neuroprotection in vivo that can then be leveraged to develop new therapeutic approaches. Unlike mammals zebrafish RGCs are resilient to injury and this study characterizes zebrafish retinal ganglion cell injury response to optic nerve transection ONT using isl2b:eGFP transgenic fish line single cell and bulk RNA sequencing and in situ hybridization. We demonstrate that zebrafish RGCs do not show subtype specific resilience to injury but show a distinct temporal injury response which includes an increase in subtype 3 a cell population having progenitor and regenerative identity. Overall design: Nine bulk RNA seq libraries were prepared from FACS purified retinal ganglion cells RGC from pooled tissue from nine sets of adult zebrafish: three libraries from injured tissue post optic nerve transection at each of 3 timepoints: 1 5 and 7 xxx post injury dpi and three from uninjured tissue on the same days.       adult zebrafish retinal ganglion cells day 1 uninjured fish set #1 GSM8691371   source name:retina|tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:uninjured|time:day 1|geo loc name:missing|collection date:missing adult zebrafish retinal ganglion cells day 1 uninjured fish set #1 Raw read data were imported to the CLC Genomics Workbench Qiagen Digital Insights licensed through the Molecular Biology Information Service of the Health Sciences Library System at the University of Pittsburgh. Differential gene expression analysis was performed on Injured versus Uninjured groups post genes with TPM=0 in one or more replicates were excluded. DEGs from 1 5 and 7 dpi time points were identified using the following filter: the maximum of the average group RPKM value >1.5 absolute fold change >1.5 FDR P value <0.05. Pathway enrichment analyses were performed using DAVID Bioinformatics Resources 6.8 https://david.ncifcrf.gov and using the KEGG database. Assembly: Danio rerio reference genome and annotations from GRCz11 from Ensembl version 106 Supplementary files format and content: Tab delimited BulkRNAseq raw counts.tsv file has raw gene counts for each Sample retina Optic nerve transection ONT was performed under dissecting microscope. The optic nerve in the left eye was severed while the right eye became the uninjured sham control. 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc. isl2b:GFP transgenic zebrafish Danio rerio used in this study were 3 6 maintained on a 14:10 hour light:dark cycle at 28C. tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:uninjured|time:day 1 GSM8691371 GSM8691371: adult zebrafish retinal ganglion cells day 1 uninjured fish set #1; Danio rerio; RNA Seq GSM8691371 r1 GSM8691371 1 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 2000   SRP552464     1DPI_uninjured_01_S16_R1_001.fastq.gz 1DPI_uninjured_01_S16_R2_001.fastq.gz fastq fastq 6400897196.0 33022714.0 GSM8691371 r1 0:96.92 1:96.91 A:1846404518;C:1284124731;G:1356841105;T:1911572894;N:1953948 96 96     1846404518 1284124731 1356841105 1911572894 1953948 SRX27130586 SRS23587590 SRA2037426 Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin                       B B biological fallback assumption illumina nextseq_v2 unknown cdna_unspecified nextera bulk bulk bulk   United States 2024-12-18 Adult Adult Eye Sensory System
34462 34462 SRR31768876 SRX27130585 SRS23587589 SRP552464 PRJNA1200121 Single Cell Profiling of Zebrafish Retinal Ganglion Cells Reveal an Injury Response Mediated by Cell Dedifferentiation and Leukocytes [RNA Seq] GSE284728 Transcriptome Analysis Retinal ganglion cells RGCs are the sole projection neurons connecting the retina to the brain and therefore play a critical role in vision. Death of RGCs during glaucoma results in irreversible loss of vision as RGCs do not xxx post injury in the human eye. There are no FDA approved drugs to prevent RGC death and/or promote RGC xxx post injury. There is a critical need to better understand the molecular underpinnings of neuroprotection in vivo that can then be leveraged to develop new therapeutic approaches. Unlike mammals zebrafish RGCs are resilient to injury and this study characterizes zebrafish retinal ganglion cell injury response to optic nerve transection ONT using isl2b:eGFP transgenic fish line single cell and bulk RNA sequencing and in situ hybridization. We demonstrate that zebrafish RGCs do not show subtype specific resilience to injury but show a distinct temporal injury response which includes an increase in subtype 3 a cell population having progenitor and regenerative identity. Overall design: Nine bulk RNA seq libraries were prepared from FACS purified retinal ganglion cells RGC from pooled tissue from nine sets of adult zebrafish: three libraries from injured tissue post optic nerve transection at each of 3 timepoints: 1 5 and 7 xxx post injury dpi and three from uninjured tissue on the same days.       adult zebrafish retinal ganglion cells 1 xxx post injury fish set #3 GSM8691370   source name:retina|tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:optic nerve transection|time:day xxx post injury|geo loc name:missing|collection date:missing adult zebrafish retinal ganglion cells 1 xxx post injury fish set #3 Raw read data were imported to the CLC Genomics Workbench Qiagen Digital Insights licensed through the Molecular Biology Information Service of the Health Sciences Library System at the University of Pittsburgh. Differential gene expression analysis was performed on Injured versus Uninjured groups post genes with TPM=0 in one or more replicates were excluded. DEGs from 1 5 and 7 dpi time points were identified using the following filter: the maximum of the average group RPKM value >1.5 absolute fold change >1.5 FDR P value <0.05. Pathway enrichment analyses were performed using DAVID Bioinformatics Resources 6.8 https://david.ncifcrf.gov and using the KEGG database. Assembly: Danio rerio reference genome and annotations from GRCz11 from Ensembl version 106 Supplementary files format and content: Tab delimited BulkRNAseq raw counts.tsv file has raw gene counts for each Sample retina Optic nerve transection ONT was performed under dissecting microscope. The optic nerve in the left eye was severed while the right eye became the uninjured sham control. 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc. isl2b:GFP transgenic zebrafish Danio rerio used in this study were 3 6 maintained on a 14:10 hour light:dark cycle at 28C. tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:optic nerve transection|time:day xxx post injury GSM8691370 GSM8691370: adult zebrafish retinal ganglion cells 1 xxx post injury fish set #3; Danio rerio; RNA Seq GSM8691370 r1 GSM8691370 1 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 2000   SRP552464     1DPI_injured_03_S15_R1_001.fastq.gz 1DPI_injured_03_S15_R2_001.fastq.gz fastq fastq 5946459133.0 30587969.0 GSM8691370 r1 0:97.20 1:97.21 A:1716543360;C:1195543685;G:1259830252;T:1772896957;N:1644879 97 97     1716543360 1195543685 1259830252 1772896957 1644879 SRX27130585 SRS23587589 SRA2037426 Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin                       B B biological fallback assumption illumina nextseq_v2 unknown cdna_unspecified nextera bulk bulk bulk   United States 2024-12-18 Adult Adult Eye Sensory System
34463 34463 SRR31768877 SRX27130584 SRS23587588 SRP552464 PRJNA1200121 Single Cell Profiling of Zebrafish Retinal Ganglion Cells Reveal an Injury Response Mediated by Cell Dedifferentiation and Leukocytes [RNA Seq] GSE284728 Transcriptome Analysis Retinal ganglion cells RGCs are the sole projection neurons connecting the retina to the brain and therefore play a critical role in vision. Death of RGCs during glaucoma results in irreversible loss of vision as RGCs do not xxx post injury in the human eye. There are no FDA approved drugs to prevent RGC death and/or promote RGC xxx post injury. There is a critical need to better understand the molecular underpinnings of neuroprotection in vivo that can then be leveraged to develop new therapeutic approaches. Unlike mammals zebrafish RGCs are resilient to injury and this study characterizes zebrafish retinal ganglion cell injury response to optic nerve transection ONT using isl2b:eGFP transgenic fish line single cell and bulk RNA sequencing and in situ hybridization. We demonstrate that zebrafish RGCs do not show subtype specific resilience to injury but show a distinct temporal injury response which includes an increase in subtype 3 a cell population having progenitor and regenerative identity. Overall design: Nine bulk RNA seq libraries were prepared from FACS purified retinal ganglion cells RGC from pooled tissue from nine sets of adult zebrafish: three libraries from injured tissue post optic nerve transection at each of 3 timepoints: 1 5 and 7 xxx post injury dpi and three from uninjured tissue on the same days.       adult zebrafish retinal ganglion cells 1 xxx post injury fish set #2 GSM8691369   source name:retina|tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:optic nerve transection|time:day xxx post injury|geo loc name:missing|collection date:missing adult zebrafish retinal ganglion cells 1 xxx post injury fish set #2 Raw read data were imported to the CLC Genomics Workbench Qiagen Digital Insights licensed through the Molecular Biology Information Service of the Health Sciences Library System at the University of Pittsburgh. Differential gene expression analysis was performed on Injured versus Uninjured groups post genes with TPM=0 in one or more replicates were excluded. DEGs from 1 5 and 7 dpi time points were identified using the following filter: the maximum of the average group RPKM value >1.5 absolute fold change >1.5 FDR P value <0.05. Pathway enrichment analyses were performed using DAVID Bioinformatics Resources 6.8 https://david.ncifcrf.gov and using the KEGG database. Assembly: Danio rerio reference genome and annotations from GRCz11 from Ensembl version 106 Supplementary files format and content: Tab delimited BulkRNAseq raw counts.tsv file has raw gene counts for each Sample retina Optic nerve transection ONT was performed under dissecting microscope. The optic nerve in the left eye was severed while the right eye became the uninjured sham control. 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc. isl2b:GFP transgenic zebrafish Danio rerio used in this study were 3 6 maintained on a 14:10 hour light:dark cycle at 28C. tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:optic nerve transection|time:day xxx post injury GSM8691369 GSM8691369: adult zebrafish retinal ganglion cells 1 xxx post injury fish set #2; Danio rerio; RNA Seq GSM8691369 r1 GSM8691369 1 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 2000   SRP552464     1DPI_injured_02_S14_R1_001.fastq.gz 1DPI_injured_02_S14_R2_001.fastq.gz fastq fastq 6366392345.0 32994721.0 GSM8691369 r1 0:96.48 1:96.47 A:1861203729;C:1243094742;G:1336477656;T:1923087937;N:2528281 96 96     1861203729 1243094742 1336477656 1923087937 2528281 SRX27130584 SRS23587588 SRA2037426 Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin                       B B biological fallback assumption illumina nextseq_v2 unknown cdna_unspecified nextera bulk bulk bulk   United States 2024-12-18 Adult Adult Eye Sensory System
34464 34464 SRR31768878 SRX27130583 SRS23587587 SRP552464 PRJNA1200121 Single Cell Profiling of Zebrafish Retinal Ganglion Cells Reveal an Injury Response Mediated by Cell Dedifferentiation and Leukocytes [RNA Seq] GSE284728 Transcriptome Analysis Retinal ganglion cells RGCs are the sole projection neurons connecting the retina to the brain and therefore play a critical role in vision. Death of RGCs during glaucoma results in irreversible loss of vision as RGCs do not xxx post injury in the human eye. There are no FDA approved drugs to prevent RGC death and/or promote RGC xxx post injury. There is a critical need to better understand the molecular underpinnings of neuroprotection in vivo that can then be leveraged to develop new therapeutic approaches. Unlike mammals zebrafish RGCs are resilient to injury and this study characterizes zebrafish retinal ganglion cell injury response to optic nerve transection ONT using isl2b:eGFP transgenic fish line single cell and bulk RNA sequencing and in situ hybridization. We demonstrate that zebrafish RGCs do not show subtype specific resilience to injury but show a distinct temporal injury response which includes an increase in subtype 3 a cell population having progenitor and regenerative identity. Overall design: Nine bulk RNA seq libraries were prepared from FACS purified retinal ganglion cells RGC from pooled tissue from nine sets of adult zebrafish: three libraries from injured tissue post optic nerve transection at each of 3 timepoints: 1 5 and 7 xxx post injury dpi and three from uninjured tissue on the same days.       adult zebrafish retinal ganglion cells 1 xxx post injury fish set #1 GSM8691368   source name:retina|tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:optic nerve transection|time:day xxx post injury|geo loc name:missing|collection date:missing adult zebrafish retinal ganglion cells 1 xxx post injury fish set #1 Raw read data were imported to the CLC Genomics Workbench Qiagen Digital Insights licensed through the Molecular Biology Information Service of the Health Sciences Library System at the University of Pittsburgh. Differential gene expression analysis was performed on Injured versus Uninjured groups post genes with TPM=0 in one or more replicates were excluded. DEGs from 1 5 and 7 dpi time points were identified using the following filter: the maximum of the average group RPKM value >1.5 absolute fold change >1.5 FDR P value <0.05. Pathway enrichment analyses were performed using DAVID Bioinformatics Resources 6.8 https://david.ncifcrf.gov and using the KEGG database. Assembly: Danio rerio reference genome and annotations from GRCz11 from Ensembl version 106 Supplementary files format and content: Tab delimited BulkRNAseq raw counts.tsv file has raw gene counts for each Sample retina Optic nerve transection ONT was performed under dissecting microscope. The optic nerve in the left eye was severed while the right eye became the uninjured sham control. 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc. isl2b:GFP transgenic zebrafish Danio rerio used in this study were 3 6 maintained on a 14:10 hour light:dark cycle at 28C. tissue:retina|cell line:adult retinal ganglion cells from isl2b:eGFP transgenic fish line|cell type:retinal ganglion cells|genotype:isl2b:eGFP transgenic fish line|treatment:optic nerve transection|time:day xxx post injury GSM8691368 GSM8691368: adult zebrafish retinal ganglion cells 1 xxx post injury fish set #1; Danio rerio; RNA Seq GSM8691368 r1 GSM8691368 1 1 5 and 7 xxx post injury dpi the retina were harvested cells dissociated using papain digestion and RGCs were FACS isolated into SmartSeq HT lysis buffer Takara Bio for mRNA extraction. cDNA sequencing libraries were prepared using a SmartSeq HT kit Takara Bio and Illumina Nextera XT kit Illumina Inc.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 2000   SRP552464     1DPI_injured_01_S13_R1_001.fastq.gz 1DPI_injured_01_S13_R2_001.fastq.gz fastq fastq 6378025205.0 33385333.0 GSM8691368 r1 0:95.51 1:95.53 A:1875735808;C:1227743564;G:1334467564;T:1936726852;N:3351417 95 95     1875735808 1227743564 1334467564 1936726852 3351417 SRX27130583 SRS23587587 SRA2037426 Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin                       B B biological fallback assumption illumina nextseq_v2 unknown cdna_unspecified nextera bulk bulk bulk   United States 2024-12-18 Adult Adult Eye Sensory System
53513 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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
61643 61643 SRR12899696 SRX9364743 SRS7586958 SRP288615 PRJNA672100 Nrl dependent and independent rod photoreceptor development in zebrafish [RNA Seq] GSE160138 Transcriptome Analysis NRL Neural retina leucine zipper is a key regulator of the fate determination and gene expression of rod photoreceptors in the retina of many vertebrates. In this study we observed a unique retinal phenotype in the nrl knockout zebrafish model characterized by reduced rods with shortened outer segments and gradually increased green cones in maturity. By tracing and comparing the developmental processes of WT and nrl knockout rods we found there might be two waves of rod genesis distinguished as nrl dependent and independent with different starting times. To reveal the underlying cellular and molecular mechanisms bulk and single cell RNA seq were performed. The changes in gene expression and cell proportion of rods and cones agreed well with our histological and immunofluorescence studies. Interestingly we found that rods exhibited noticeable heterogeneities in the gene expression patterns and a part of rods in nrl knockout zebrafish misexpressed the green cone genes reflecting a hybrid status of rod and green cone. Furthermore we identified mafba as a novel regulator of rod genesis which was responsible for the development of rods in nrl knockout zebrafish. Our study will largely improve the current understanding of the developmental processes and regulatory mechanisms of rods in zebrafish and probably other species and may facilitate future studies in the fields of retinal development and retinal degeneration. Overall design: Two WT and two nrl KO samples two biological replicates were analyzed. Each sample represented a pool of RNAs from three zebrafish retinas at the age of 2 month. The differentially expressed genes between WT and nrl KO groups were identified. parent bioproject:PRJNA672094 pubmed:35245286   nrl R2 GSM4861209   source name:Retina|tissue:Retina|strain:AB|age:2 mpf KO nrl R2 Quality control mapping and expression analysis: Image analysis and base calling were conducted by the HiSeq Control Software HCS + OLB + GAPipeline 1.6 Illumina on the HiSeq instrument. The high quality clean data were generated by Trimmomatic v0.30 and aligned to the reference genome of zebrafish via software Hisat2 v2.0.1. Differential expression analysis: Gene and isoform expression levels were estimated by HTSeq v0.6.1. Differential expression analysis was performed by DESeq Bioconductor package. post adjusted by Benjamini and Hochberg’s approach for controlling the false discovery rate the threshold of p values was set to <0.05 to detect differentially expressed genes. GO and KEGG enrichment analysis: GO TermFinder was used identifying Gene Ontology GO terms that annotate a list of enriched genes with a significant p value less than 0.05. KEGG Kyoto Encyclopedia of Genes and Genomes is a collection of databases dealing with genomes biological pathways diseases drugs and chemical substances http://en.wikipedia.org/wiki/KEGG which was used to enrich significant differential expression gene in KEGG pathways. Genome build: GRCz11 from Ensembl Supplementary files format and content: Tab delimited text files including the raw counts and FPKM values for each Sample Supplementary files format and content: Tab delimited text files showing the foldchanges and p values of the differentially expressed genes Retina   For each RNA sample three eyes lens removed from different zebrafish were homogenized in RNAiso Plus reagent Takara Cat# 9108. Total RNA was extracted following the operation manual. The total RNA samples were quantified and qualified by Agilent 2100 Bioanalyzer Agilent Technologies and NanoDrop Thermo Fisher Scientific. RNA samples with RIN ≥9.5 and A260/280 ≥1.9 were used for RNA seq. The preparations of next generation sequencing library were constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina according to the manufacturer’s protocol.   tissue:Retina|strain:AB|age:2 mpf KO GSM4861209 GSM4861209: nrl R2; Danio rerio; RNA Seq GSM4861209   1 For each RNA sample three eyes lens removed from different zebrafish were homogenized in RNAiso Plus reagent Takara Cat# 9108. Total RNA was extracted following the operation manual. The total RNA samples were quantified and qualified by Agilent 2100 Bioanalyzer Agilent Technologies and NanoDrop Thermo Fisher Scientific. RNA samples with RIN ≥9.5 and A260/280 ≥1.9 were used for RNA seq. The preparations of next generation sequencing library were constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina according to the manufacturer's protocol. GEO Accession:GSM4861209 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   SRP288615     nrl-R2_combined_R1.fastq.gz nrl-R2_combined_R2.fastq.gz fastq fastq 7231497600.0 24104992.0 GSM4861209 r1 0:150 1:150 A:1995457626;C:1613822880;G:1630032692;T:1991951852;N:232550 150 150     1995457626 1613822880 1630032692 1991951852 232550 SRX9364743 SRS7586958 SRA1147909 GEO College of Life Science and Technology, Huazhong University of Science and Technology 2 0.91539 0.91383 0.13441 0.13499 0.69296 0.70021 0.5139 0.50426 150 150 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nebnext sc_generic bulk bulk   China 2020-10-26 Juvenile Juvenile Eye Sensory System
61644 61644 SRR12899695 SRX9364742 SRS7586957 SRP288615 PRJNA672100 Nrl dependent and independent rod photoreceptor development in zebrafish [RNA Seq] GSE160138 Transcriptome Analysis NRL Neural retina leucine zipper is a key regulator of the fate determination and gene expression of rod photoreceptors in the retina of many vertebrates. In this study we observed a unique retinal phenotype in the nrl knockout zebrafish model characterized by reduced rods with shortened outer segments and gradually increased green cones in maturity. By tracing and comparing the developmental processes of WT and nrl knockout rods we found there might be two waves of rod genesis distinguished as nrl dependent and independent with different starting times. To reveal the underlying cellular and molecular mechanisms bulk and single cell RNA seq were performed. The changes in gene expression and cell proportion of rods and cones agreed well with our histological and immunofluorescence studies. Interestingly we found that rods exhibited noticeable heterogeneities in the gene expression patterns and a part of rods in nrl knockout zebrafish misexpressed the green cone genes reflecting a hybrid status of rod and green cone. Furthermore we identified mafba as a novel regulator of rod genesis which was responsible for the development of rods in nrl knockout zebrafish. Our study will largely improve the current understanding of the developmental processes and regulatory mechanisms of rods in zebrafish and probably other species and may facilitate future studies in the fields of retinal development and retinal degeneration. Overall design: Two WT and two nrl KO samples two biological replicates were analyzed. Each sample represented a pool of RNAs from three zebrafish retinas at the age of 2 month. The differentially expressed genes between WT and nrl KO groups were identified. parent bioproject:PRJNA672094 pubmed:35245286   nrl R1 GSM4861208   source name:Retina|tissue:Retina|strain:AB|age:2 mpf KO nrl R1 Quality control mapping and expression analysis: Image analysis and base calling were conducted by the HiSeq Control Software HCS + OLB + GAPipeline 1.6 Illumina on the HiSeq instrument. The high quality clean data were generated by Trimmomatic v0.30 and aligned to the reference genome of zebrafish via software Hisat2 v2.0.1. Differential expression analysis: Gene and isoform expression levels were estimated by HTSeq v0.6.1. Differential expression analysis was performed by DESeq Bioconductor package. post adjusted by Benjamini and Hochberg’s approach for controlling the false discovery rate the threshold of p values was set to <0.05 to detect differentially expressed genes. GO and KEGG enrichment analysis: GO TermFinder was used identifying Gene Ontology GO terms that annotate a list of enriched genes with a significant p value less than 0.05. KEGG Kyoto Encyclopedia of Genes and Genomes is a collection of databases dealing with genomes biological pathways diseases drugs and chemical substances http://en.wikipedia.org/wiki/KEGG which was used to enrich significant differential expression gene in KEGG pathways. Genome build: GRCz11 from Ensembl Supplementary files format and content: Tab delimited text files including the raw counts and FPKM values for each Sample Supplementary files format and content: Tab delimited text files showing the foldchanges and p values of the differentially expressed genes Retina   For each RNA sample three eyes lens removed from different zebrafish were homogenized in RNAiso Plus reagent Takara Cat# 9108. Total RNA was extracted following the operation manual. The total RNA samples were quantified and qualified by Agilent 2100 Bioanalyzer Agilent Technologies and NanoDrop Thermo Fisher Scientific. RNA samples with RIN ≥9.5 and A260/280 ≥1.9 were used for RNA seq. The preparations of next generation sequencing library were constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina according to the manufacturer’s protocol.   tissue:Retina|strain:AB|age:2 mpf KO GSM4861208 GSM4861208: nrl R1; Danio rerio; RNA Seq GSM4861208   1 For each RNA sample three eyes lens removed from different zebrafish were homogenized in RNAiso Plus reagent Takara Cat# 9108. Total RNA was extracted following the operation manual. The total RNA samples were quantified and qualified by Agilent 2100 Bioanalyzer Agilent Technologies and NanoDrop Thermo Fisher Scientific. RNA samples with RIN ≥9.5 and A260/280 ≥1.9 were used for RNA seq. The preparations of next generation sequencing library were constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina according to the manufacturer's protocol. GEO Accession:GSM4861208 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   SRP288615     nrl-R1_combined_R1.fastq.gz nrl-R1_combined_R2.fastq.gz fastq fastq 6989998200.0 23299994.0 GSM4861208 r1 0:150 1:150 A:1947895730;C:1539849390;G:1551787456;T:1950215842;N:249782 150 150     1947895730 1539849390 1551787456 1950215842 249782 SRX9364742 SRS7586957 SRA1147909 GEO College of Life Science and Technology, Huazhong University of Science and Technology 2 0.90616 0.9059 0.15209 0.15341 0.68998 0.69885 0.49879 0.49931 150 150 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nebnext sc_generic bulk bulk   China 2020-10-26 Juvenile Juvenile Eye Sensory System
61645 61645 SRR12899694 SRX9364741 SRS7586956 SRP288615 PRJNA672100 Nrl dependent and independent rod photoreceptor development in zebrafish [RNA Seq] GSE160138 Transcriptome Analysis NRL Neural retina leucine zipper is a key regulator of the fate determination and gene expression of rod photoreceptors in the retina of many vertebrates. In this study we observed a unique retinal phenotype in the nrl knockout zebrafish model characterized by reduced rods with shortened outer segments and gradually increased green cones in maturity. By tracing and comparing the developmental processes of WT and nrl knockout rods we found there might be two waves of rod genesis distinguished as nrl dependent and independent with different starting times. To reveal the underlying cellular and molecular mechanisms bulk and single cell RNA seq were performed. The changes in gene expression and cell proportion of rods and cones agreed well with our histological and immunofluorescence studies. Interestingly we found that rods exhibited noticeable heterogeneities in the gene expression patterns and a part of rods in nrl knockout zebrafish misexpressed the green cone genes reflecting a hybrid status of rod and green cone. Furthermore we identified mafba as a novel regulator of rod genesis which was responsible for the development of rods in nrl knockout zebrafish. Our study will largely improve the current understanding of the developmental processes and regulatory mechanisms of rods in zebrafish and probably other species and may facilitate future studies in the fields of retinal development and retinal degeneration. Overall design: Two WT and two nrl KO samples two biological replicates were analyzed. Each sample represented a pool of RNAs from three zebrafish retinas at the age of 2 month. The differentially expressed genes between WT and nrl KO groups were identified. parent bioproject:PRJNA672094 pubmed:35245286   WT R2 GSM4861207   source name:Retina|tissue:Retina|strain:AB|age:2 mpf type WT R2 Quality control mapping and expression analysis: Image analysis and base calling were conducted by the HiSeq Control Software HCS + OLB + GAPipeline 1.6 Illumina on the HiSeq instrument. The high quality clean data were generated by Trimmomatic v0.30 and aligned to the reference genome of zebrafish via software Hisat2 v2.0.1. Differential expression analysis: Gene and isoform expression levels were estimated by HTSeq v0.6.1. Differential expression analysis was performed by DESeq Bioconductor package. post adjusted by Benjamini and Hochberg’s approach for controlling the false discovery rate the threshold of p values was set to <0.05 to detect differentially expressed genes. GO and KEGG enrichment analysis: GO TermFinder was used identifying Gene Ontology GO terms that annotate a list of enriched genes with a significant p value less than 0.05. KEGG Kyoto Encyclopedia of Genes and Genomes is a collection of databases dealing with genomes biological pathways diseases drugs and chemical substances http://en.wikipedia.org/wiki/KEGG which was used to enrich significant differential expression gene in KEGG pathways. Genome build: GRCz11 from Ensembl Supplementary files format and content: Tab delimited text files including the raw counts and FPKM values for each Sample Supplementary files format and content: Tab delimited text files showing the foldchanges and p values of the differentially expressed genes Retina   For each RNA sample three eyes lens removed from different zebrafish were homogenized in RNAiso Plus reagent Takara Cat# 9108. Total RNA was extracted following the operation manual. The total RNA samples were quantified and qualified by Agilent 2100 Bioanalyzer Agilent Technologies and NanoDrop Thermo Fisher Scientific. RNA samples with RIN ≥9.5 and A260/280 ≥1.9 were used for RNA seq. The preparations of next generation sequencing library were constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina according to the manufacturer’s protocol.   tissue:Retina|strain:AB|age:2 mpf type GSM4861207 GSM4861207: WT R2; Danio rerio; RNA Seq GSM4861207   1 For each RNA sample three eyes lens removed from different zebrafish were homogenized in RNAiso Plus reagent Takara Cat# 9108. Total RNA was extracted following the operation manual. The total RNA samples were quantified and qualified by Agilent 2100 Bioanalyzer Agilent Technologies and NanoDrop Thermo Fisher Scientific. RNA samples with RIN ≥9.5 and A260/280 ≥1.9 were used for RNA seq. The preparations of next generation sequencing library were constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina according to the manufacturer's protocol. GEO Accession:GSM4861207 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   SRP288615     WT-R2_combined_R1.fastq.gz WT-R2_combined_R2.fastq.gz fastq fastq 7066558200.0 23555194.0 GSM4861207 r1 0:150 1:150 A:1911018201;C:1615965707;G:1630234641;T:1909071420;N:268231 150 150     1911018201 1615965707 1630234641 1909071420 268231 SRX9364741 SRS7586956 SRA1147909 GEO College of Life Science and Technology, Huazhong University of Science and Technology 2 0.91673 0.91647 0.11542 0.11684 0.68781 0.69637 0.48784 0.49134 150 150 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nebnext sc_generic bulk bulk   China 2020-10-26 Juvenile Juvenile Eye Sensory System
61646 61646 SRR12899693 SRX9364740 SRS7586955 SRP288615 PRJNA672100 Nrl dependent and independent rod photoreceptor development in zebrafish [RNA Seq] GSE160138 Transcriptome Analysis NRL Neural retina leucine zipper is a key regulator of the fate determination and gene expression of rod photoreceptors in the retina of many vertebrates. In this study we observed a unique retinal phenotype in the nrl knockout zebrafish model characterized by reduced rods with shortened outer segments and gradually increased green cones in maturity. By tracing and comparing the developmental processes of WT and nrl knockout rods we found there might be two waves of rod genesis distinguished as nrl dependent and independent with different starting times. To reveal the underlying cellular and molecular mechanisms bulk and single cell RNA seq were performed. The changes in gene expression and cell proportion of rods and cones agreed well with our histological and immunofluorescence studies. Interestingly we found that rods exhibited noticeable heterogeneities in the gene expression patterns and a part of rods in nrl knockout zebrafish misexpressed the green cone genes reflecting a hybrid status of rod and green cone. Furthermore we identified mafba as a novel regulator of rod genesis which was responsible for the development of rods in nrl knockout zebrafish. Our study will largely improve the current understanding of the developmental processes and regulatory mechanisms of rods in zebrafish and probably other species and may facilitate future studies in the fields of retinal development and retinal degeneration. Overall design: Two WT and two nrl KO samples two biological replicates were analyzed. Each sample represented a pool of RNAs from three zebrafish retinas at the age of 2 month. The differentially expressed genes between WT and nrl KO groups were identified. parent bioproject:PRJNA672094 pubmed:35245286   WT R1 GSM4861206   source name:Retina|tissue:Retina|strain:AB|age:2 mpf type WT R1 Quality control mapping and expression analysis: Image analysis and base calling were conducted by the HiSeq Control Software HCS + OLB + GAPipeline 1.6 Illumina on the HiSeq instrument. The high quality clean data were generated by Trimmomatic v0.30 and aligned to the reference genome of zebrafish via software Hisat2 v2.0.1. Differential expression analysis: Gene and isoform expression levels were estimated by HTSeq v0.6.1. Differential expression analysis was performed by DESeq Bioconductor package. post adjusted by Benjamini and Hochberg’s approach for controlling the false discovery rate the threshold of p values was set to <0.05 to detect differentially expressed genes. GO and KEGG enrichment analysis: GO TermFinder was used identifying Gene Ontology GO terms that annotate a list of enriched genes with a significant p value less than 0.05. KEGG Kyoto Encyclopedia of Genes and Genomes is a collection of databases dealing with genomes biological pathways diseases drugs and chemical substances http://en.wikipedia.org/wiki/KEGG which was used to enrich significant differential expression gene in KEGG pathways. Genome build: GRCz11 from Ensembl Supplementary files format and content: Tab delimited text files including the raw counts and FPKM values for each Sample Supplementary files format and content: Tab delimited text files showing the foldchanges and p values of the differentially expressed genes Retina   For each RNA sample three eyes lens removed from different zebrafish were homogenized in RNAiso Plus reagent Takara Cat# 9108. Total RNA was extracted following the operation manual. The total RNA samples were quantified and qualified by Agilent 2100 Bioanalyzer Agilent Technologies and NanoDrop Thermo Fisher Scientific. RNA samples with RIN ≥9.5 and A260/280 ≥1.9 were used for RNA seq. The preparations of next generation sequencing library were constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina according to the manufacturer’s protocol.   tissue:Retina|strain:AB|age:2 mpf type GSM4861206 GSM4861206: WT R1; Danio rerio; RNA Seq GSM4861206   1 For each RNA sample three eyes lens removed from different zebrafish were homogenized in RNAiso Plus reagent Takara Cat# 9108. Total RNA was extracted following the operation manual. The total RNA samples were quantified and qualified by Agilent 2100 Bioanalyzer Agilent Technologies and NanoDrop Thermo Fisher Scientific. RNA samples with RIN ≥9.5 and A260/280 ≥1.9 were used for RNA seq. The preparations of next generation sequencing library were constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina according to the manufacturer's protocol. GEO Accession:GSM4861206 RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   SRP288615     WT-R1_combined_R1.fastq.gz WT-R1_combined_R2.fastq.gz fastq fastq 6933407700.0 23111359.0 GSM4861206 r1 0:150 1:150 A:1917552178;C:1542500715;G:1553125711;T:1919988847;N:240249 150 150     1917552178 1542500715 1553125711 1919988847 240249 SRX9364740 SRS7586955 SRA1147909 GEO College of Life Science and Technology, Huazhong University of Science and Technology 2 0.90701 0.90456 0.14405 0.14325 0.68335 0.69221 0.49207 0.4926 150 150 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nebnext sc_generic bulk bulk   China 2020-10-26 Juvenile Juvenile Eye Sensory System
71694 71694 SRR21966803 SRX17950306 SRS15467734 SRP403321 PRJNA891767 RNA seq of retina of zebrafish PRJNA891767 Other We performed bulk RNA sequencing on the zebrafish in an aim to identify the differential expression genes between retinal inflammation and normal group.       LPS 72hpi 1 LPS 72hpi 1   isolate:not applicable|age:6 month|sex:not applicable|tissue:retina|replicate:LPS 72hpi biological replicate1|BioSampleModel:Model organism or animal                 RNA seq of retina LPS 72hpi 1 LPS 72hpi 1 RNA Extraction library construction and sequencingTotal RNA was extracted using Trizol reagent kit Invitrogen Carlsbad CA USA according to the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer Agilent Technologies Palo Alto CA USA and checked using RNase free agarose gel electrophoresis. post total RNA was extracted eukaryotic mRNA was enriched by OligodT beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second strand cDNA were synthesized by DNA polymerase I RNase H dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kitQiagen Venlo The Netherlands end repaired polyA added and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis PCR amplified and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. Guangzhou China.     RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP403321     LPS_72hpi_1_1.fq.gz LPS_72hpi_1_2.fq.gz fastq fastq 6155981100.0 20519937.0 LPS 72hpi 1 1.fq.gz 0:150 1:150 A:1710578291;C:1365661745;G:1380694358;T:1698845537;N:201169 150 150     1710578291 1365661745 1380694358 1698845537 201169 SRX17950306 SRS15467734 SRA1523113 The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital|Henan International Joint Research Laboratory for The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital 2 0.93477 0.93681 0.13209 0.13274 0.71612 0.71463 0.50765 0.50927 150 150 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk bulk bulk   China 2022-10-19 Adult Adult Eye Sensory System
71695 71695 SRR21966804 SRX17950305 SRS15467733 SRP403321 PRJNA891767 RNA seq of retina of zebrafish PRJNA891767 Other We performed bulk RNA sequencing on the zebrafish in an aim to identify the differential expression genes between retinal inflammation and normal group.       LPS 24hpi 3 LPS 24hpi 3   isolate:not applicable|age:6 month|sex:not applicable|tissue:retina|replicate:LPS 24hpi biological replicate3|BioSampleModel:Model organism or animal                 RNA seq of retina LPS 24hpi 3 LPS 24hpi 3 RNA Extraction library construction and sequencingTotal RNA was extracted using Trizol reagent kit Invitrogen Carlsbad CA USA according to the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer Agilent Technologies Palo Alto CA USA and checked using RNase free agarose gel electrophoresis. post total RNA was extracted eukaryotic mRNA was enriched by OligodT beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second strand cDNA were synthesized by DNA polymerase I RNase H dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kitQiagen Venlo The Netherlands end repaired polyA added and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis PCR amplified and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. Guangzhou China.     RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP403321     LPS_24hpi_3_1.fq.gz LPS_24hpi_3_2.fq.gz fastq fastq 5757291600.0 19190972.0 LPS 24hpi 3 1.fq.gz 0:150 1:150 A:1600505453;C:1274738155;G:1290634640;T:1591222631;N:190721 150 150     1600505453 1274738155 1290634640 1591222631 190721 SRX17950305 SRS15467733 SRA1523113 The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital|Henan International Joint Research Laboratory for The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital 2 0.93363 0.93509 0.12986 0.13001 0.7069 0.70741 0.51061 0.51114 150 150 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk bulk bulk   China 2022-10-19 Adult Adult Eye Sensory System
71696 71696 SRR21966805 SRX17950304 SRS15467732 SRP403321 PRJNA891767 RNA seq of retina of zebrafish PRJNA891767 Other We performed bulk RNA sequencing on the zebrafish in an aim to identify the differential expression genes between retinal inflammation and normal group.       LPS 24hpi 2 LPS 24hpi 2   isolate:not applicable|age:6 month|sex:not applicable|tissue:retina|replicate:LPS 24hpi biological replicate2|BioSampleModel:Model organism or animal                 RNA seq of retina LPS 24hpi 2 LPS 24hpi 2 RNA Extraction library construction and sequencingTotal RNA was extracted using Trizol reagent kit Invitrogen Carlsbad CA USA according to the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer Agilent Technologies Palo Alto CA USA and checked using RNase free agarose gel electrophoresis. post total RNA was extracted eukaryotic mRNA was enriched by OligodT beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second strand cDNA were synthesized by DNA polymerase I RNase H dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kitQiagen Venlo The Netherlands end repaired polyA added and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis PCR amplified and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. Guangzhou China.     RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP403321     LPS_24hpi_2_1.fq.gz LPS_24hpi_2_2.fq.gz fastq fastq 8088450600.0 26961502.0 LPS 24hpi 2 1.fq.gz 0:150 1:150 A:2253499351;C:1789037027;G:1805352149;T:2240301026;N:261047 150 150     2253499351 1789037027 1805352149 2240301026 261047 SRX17950304 SRS15467732 SRA1523113 The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital|Henan International Joint Research Laboratory for The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital 2 0.93055 0.93268 0.13977 0.1409 0.71161 0.71035 0.50046 0.50273 150 150 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk bulk bulk   China 2022-10-19 Adult Adult Eye Sensory System
71697 71697 SRR21966806 SRX17950303 SRS15467731 SRP403321 PRJNA891767 RNA seq of retina of zebrafish PRJNA891767 Other We performed bulk RNA sequencing on the zebrafish in an aim to identify the differential expression genes between retinal inflammation and normal group.       LPS 24hpi 1 LPS 24hpi 1   isolate:not applicable|age:6 month|sex:not applicable|tissue:retina|replicate:LPS 24hpi biological replicate1|BioSampleModel:Model organism or animal                 RNA seq of retina LPS 24hpi 1 LPS 24hpi 1 RNA Extraction library construction and sequencingTotal RNA was extracted using Trizol reagent kit Invitrogen Carlsbad CA USA according to the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer Agilent Technologies Palo Alto CA USA and checked using RNase free agarose gel electrophoresis. post total RNA was extracted eukaryotic mRNA was enriched by OligodT beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second strand cDNA were synthesized by DNA polymerase I RNase H dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kitQiagen Venlo The Netherlands end repaired polyA added and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis PCR amplified and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. Guangzhou China.     RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP403321     LPS_24hpi_1_1.fq.gz LPS_24hpi_1_2.fq.gz fastq fastq 7708943100.0 25696477.0 LPS 24hpi 1 1.fq.gz 0:150 1:150 A:2152004377;C:1700188663;G:1714406482;T:2142095710;N:247868 150 150     2152004377 1700188663 1714406482 2142095710 247868 SRX17950303 SRS15467731 SRA1523113 The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital|Henan International Joint Research Laboratory for The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital 2 0.93249 0.93531 0.14198 0.14312 0.70999 0.70936 0.50462 0.51594 150 150 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk bulk bulk   China 2022-10-19 Adult Adult Eye Sensory System
71698 71698 SRR21966807 SRX17950302 SRS15467730 SRP403321 PRJNA891767 RNA seq of retina of zebrafish PRJNA891767 Other We performed bulk RNA sequencing on the zebrafish in an aim to identify the differential expression genes between retinal inflammation and normal group.       LPS 12hpi 3 LPS 12hpi 3   isolate:not applicable|age:6 month|sex:not applicable|tissue:retina|replicate:LPS 12hpi biological replicate3|BioSampleModel:Model organism or animal                 RNA seq of retina LPS 12hpi 3 LPS 12hpi 3 RNA Extraction library construction and sequencingTotal RNA was extracted using Trizol reagent kit Invitrogen Carlsbad CA USA according to the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer Agilent Technologies Palo Alto CA USA and checked using RNase free agarose gel electrophoresis. post total RNA was extracted eukaryotic mRNA was enriched by OligodT beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second strand cDNA were synthesized by DNA polymerase I RNase H dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kitQiagen Venlo The Netherlands end repaired polyA added and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis PCR amplified and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. Guangzhou China.     RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP403321     LPS_12hpi_3_1.fq.gz LPS_12hpi_3_2.fq.gz fastq fastq 8489127300.0 28297091.0 LPS 12hpi 3 1.fq.gz 0:150 1:150 A:2357037165;C:1883515307;G:1904547446;T:2343748318;N:279064 150 150     2357037165 1883515307 1904547446 2343748318 279064 SRX17950302 SRS15467730 SRA1523113 The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital|Henan International Joint Research Laboratory for The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital 2 0.93425 0.93458 0.13346 0.13336 0.71104 0.71031 0.49092 0.49094 150 150 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk bulk bulk   China 2022-10-19 Adult Adult Eye Sensory System
71699 71699 SRR21966808 SRX17950301 SRS15467729 SRP403321 PRJNA891767 RNA seq of retina of zebrafish PRJNA891767 Other We performed bulk RNA sequencing on the zebrafish in an aim to identify the differential expression genes between retinal inflammation and normal group.       LPS 12hpi 2 LPS 12hpi 2   isolate:not applicable|age:6 month|sex:not applicable|tissue:retina|replicate:LPS 12hpi biological replicate2|BioSampleModel:Model organism or animal                 RNA seq of retina LPS 12hpi 2 LPS 12hpi 2 RNA Extraction library construction and sequencingTotal RNA was extracted using Trizol reagent kit Invitrogen Carlsbad CA USA according to the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer Agilent Technologies Palo Alto CA USA and checked using RNase free agarose gel electrophoresis. post total RNA was extracted eukaryotic mRNA was enriched by OligodT beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second strand cDNA were synthesized by DNA polymerase I RNase H dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kitQiagen Venlo The Netherlands end repaired polyA added and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis PCR amplified and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. Guangzhou China.     RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP403321     LPS_12hpi_2_1.fq.gz LPS_12hpi_2_2.fq.gz fastq fastq 5516109300.0 18387031.0 LPS 12hpi 2 1.fq.gz 0:150 1:150 A:1536002301;C:1218719690;G:1233544971;T:1527663175;N:179163 150 150     1536002301 1218719690 1233544971 1527663175 179163 SRX17950301 SRS15467729 SRA1523113 The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital|Henan International Joint Research Laboratory for The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital 2 0.93195 0.9332 0.13229 0.13195 0.7091 0.70997 0.5012 0.49629 150 150 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk bulk bulk   China 2022-10-19 Adult Adult Eye Sensory System
71700 71700 SRR21966809 SRX17950300 SRS15467728 SRP403321 PRJNA891767 RNA seq of retina of zebrafish PRJNA891767 Other We performed bulk RNA sequencing on the zebrafish in an aim to identify the differential expression genes between retinal inflammation and normal group.       LPS 12hpi 1 LPS 12hpi 1   isolate:not applicable|age:6 month|sex:not applicable|tissue:retina|replicate:LPS 12hpi biological replicate1|BioSampleModel:Model organism or animal                 RNA seq of retina LPS 12hpi 1 LPS 12hpi 1 RNA Extraction library construction and sequencingTotal RNA was extracted using Trizol reagent kit Invitrogen Carlsbad CA USA according to the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer Agilent Technologies Palo Alto CA USA and checked using RNase free agarose gel electrophoresis. post total RNA was extracted eukaryotic mRNA was enriched by OligodT beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second strand cDNA were synthesized by DNA polymerase I RNase H dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kitQiagen Venlo The Netherlands end repaired polyA added and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis PCR amplified and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. Guangzhou China.     RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP403321     LPS_12hpi_1_1.fq.gz LPS_12hpi_1_2.fq.gz fastq fastq 7125525300.0 23751751.0 LPS 12hpi 1 1.fq.gz 0:150 1:150 A:1985336488;C:1573087021;G:1591179821;T:1975739599;N:182371 150 150     1985336488 1573087021 1591179821 1975739599 182371 SRX17950300 SRS15467728 SRA1523113 The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital|Henan International Joint Research Laboratory for The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital 2 0.92888 0.92955 0.13091 0.13004 0.71161 0.71228 0.49857 0.49684 150 150 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk bulk bulk   China 2022-10-19 Adult Adult Eye Sensory System
71701 71701 SRR21966810 SRX17950299 SRS15467727 SRP403321 PRJNA891767 RNA seq of retina of zebrafish PRJNA891767 Other We performed bulk RNA sequencing on the zebrafish in an aim to identify the differential expression genes between retinal inflammation and normal group.       blank 3 blank 3   isolate:not applicable|age:6 month|sex:not applicable|tissue:retina|replicate:blank biological replicate3|BioSampleModel:Model organism or animal                 RNA seq of retina blank 3 blank 3 RNA Extraction library construction and sequencingTotal RNA was extracted using Trizol reagent kit Invitrogen Carlsbad CA USA according to the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer Agilent Technologies Palo Alto CA USA and checked using RNase free agarose gel electrophoresis. post total RNA was extracted eukaryotic mRNA was enriched by OligodT beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second strand cDNA were synthesized by DNA polymerase I RNase H dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kitQiagen Venlo The Netherlands end repaired polyA added and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis PCR amplified and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. Guangzhou China.     RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP403321     blank_3_1.fq.gz blank_3_2.fq.gz fastq fastq 6765601200.0 22552004.0 blank 3 1.fq.gz 0:150 1:150 A:1883448626;C:1495698262;G:1509442226;T:1876823722;N:188364 150 150     1883448626 1495698262 1509442226 1876823722 188364 SRX17950299 SRS15467727 SRA1523113 The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital|Henan International Joint Research Laboratory for The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital 2 0.93243 0.93264 0.13358 0.13349 0.71786 0.71816 0.50554 0.50668 150 150 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk bulk bulk   China 2022-10-19 Adult Adult Eye Sensory System
71702 71702 SRR21966811 SRX17950298 SRS15467726 SRP403321 PRJNA891767 RNA seq of retina of zebrafish PRJNA891767 Other We performed bulk RNA sequencing on the zebrafish in an aim to identify the differential expression genes between retinal inflammation and normal group.       blank 2 blank 2   isolate:not applicable|age:6 month|sex:not applicable|tissue:retina|replicate:blank biological replicate2|BioSampleModel:Model organism or animal                 RNA seq of retina blank 2 blank 2 RNA Extraction library construction and sequencingTotal RNA was extracted using Trizol reagent kit Invitrogen Carlsbad CA USA according to the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer Agilent Technologies Palo Alto CA USA and checked using RNase free agarose gel electrophoresis. post total RNA was extracted eukaryotic mRNA was enriched by OligodT beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second strand cDNA were synthesized by DNA polymerase I RNase H dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kitQiagen Venlo The Netherlands end repaired polyA added and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis PCR amplified and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. Guangzhou China.     RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP403321     blank_2_1.fq.gz blank_2_2.fq.gz fastq fastq 5482974000.0 18276580.0 blank 2 1.fq.gz 0:150 1:150 A:1524776662;C:1215165021;G:1225270081;T:1517587506;N:174730 150 150     1524776662 1215165021 1225270081 1517587506 174730 SRX17950298 SRS15467726 SRA1523113 The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital|Henan International Joint Research Laboratory for The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital 2 0.93468 0.93712 0.13609 0.13746 0.71954 0.7194 0.51594 0.50636 150 150 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk bulk bulk   China 2022-10-19 Adult Adult Eye Sensory System
71703 71703 SRR21966812 SRX17950297 SRS15467725 SRP403321 PRJNA891767 RNA seq of retina of zebrafish PRJNA891767 Other We performed bulk RNA sequencing on the zebrafish in an aim to identify the differential expression genes between retinal inflammation and normal group.       blank 1 blank 1   isolate:not applicable|age:6 month|sex:not applicable|tissue:retina|replicate:blank biological replicate1|BioSampleModel:Model organism or animal                 RNA seq of retina blank 1 blank 1 RNA Extraction library construction and sequencingTotal RNA was extracted using Trizol reagent kit Invitrogen Carlsbad CA USA according to the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer Agilent Technologies Palo Alto CA USA and checked using RNase free agarose gel electrophoresis. post total RNA was extracted eukaryotic mRNA was enriched by OligodT beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second strand cDNA were synthesized by DNA polymerase I RNase H dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kitQiagen Venlo The Netherlands end repaired polyA added and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis PCR amplified and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. Guangzhou China.     RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP403321     blank_1_1.fq.gz blank_1_2.fq.gz fastq fastq 6055404900.0 20184683.0 blank 1 1.fq.gz 0:150 1:150 A:1683019225;C:1345039599;G:1357544427;T:1669605645;N:196004 150 150     1683019225 1345039599 1357544427 1669605645 196004 SRX17950297 SRS15467725 SRA1523113 The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital|Henan International Joint Research Laboratory for The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital 2 0.93509 0.93705 0.13072 0.13146 0.72019 0.71918 0.51001 0.50439 150 150 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk bulk bulk   China 2022-10-19 Adult Adult Eye Sensory System
71704 71704 SRR21966813 SRX17950296 SRS15467724 SRP403321 PRJNA891767 RNA seq of retina of zebrafish PRJNA891767 Other We performed bulk RNA sequencing on the zebrafish in an aim to identify the differential expression genes between retinal inflammation and normal group.       saline 72hpi 3 saline 72hpi 3   isolate:not applicable|age:6 month|sex:not applicable|tissue:retina|replicate:saline 72hpi biological replicate3|BioSampleModel:Model organism or animal                 RNA seq of retina saline 72hpi 3 saline 72hpi 3 RNA Extraction library construction and sequencingTotal RNA was extracted using Trizol reagent kit Invitrogen Carlsbad CA USA according to the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer Agilent Technologies Palo Alto CA USA and checked using RNase free agarose gel electrophoresis. post total RNA was extracted eukaryotic mRNA was enriched by OligodT beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second strand cDNA were synthesized by DNA polymerase I RNase H dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kitQiagen Venlo The Netherlands end repaired polyA added and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis PCR amplified and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. Guangzhou China.     RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP403321     saline_72hpi_3_1.fq.gz saline_72hpi_3_2.fq.gz fastq fastq 6267068700.0 20890229.0 saline 72hpi 3 1.fq.gz 0:150 1:150 A:1752026997;C:1378943130;G:1392402638;T:1743491403;N:204532 150 150     1752026997 1378943130 1392402638 1743491403 204532 SRX17950296 SRS15467724 SRA1523113 The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital|Henan International Joint Research Laboratory for The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital 2 0.93056 0.93195 0.15033 0.1502 0.7166 0.71536 0.51533 0.51642 150 150 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk bulk bulk   China 2022-10-19 Adult Adult Eye Sensory System
71705 71705 SRR21966814 SRX17950295 SRS15467723 SRP403321 PRJNA891767 RNA seq of retina of zebrafish PRJNA891767 Other We performed bulk RNA sequencing on the zebrafish in an aim to identify the differential expression genes between retinal inflammation and normal group.       saline 72hpi 2 saline 72hpi 2   isolate:not applicable|age:6 month|sex:not applicable|tissue:retina|replicate:saline 72hpi biological replicate2|BioSampleModel:Model organism or animal                 RNA seq of retina saline 72hpi 2 saline 72hpi 2 RNA Extraction library construction and sequencingTotal RNA was extracted using Trizol reagent kit Invitrogen Carlsbad CA USA according to the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer Agilent Technologies Palo Alto CA USA and checked using RNase free agarose gel electrophoresis. post total RNA was extracted eukaryotic mRNA was enriched by OligodT beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second strand cDNA were synthesized by DNA polymerase I RNase H dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kitQiagen Venlo The Netherlands end repaired polyA added and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis PCR amplified and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. Guangzhou China.     RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP403321     saline_72hpi_2_1.fq.gz saline_72hpi_2_2.fq.gz fastq fastq 5842822800.0 19476076.0 saline 72hpi 2 1.fq.gz 0:150 1:150 A:1636978240;C:1282810268;G:1294782269;T:1628060178;N:191845 150 150     1636978240 1282810268 1294782269 1628060178 191845 SRX17950295 SRS15467723 SRA1523113 The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital|Henan International Joint Research Laboratory for The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital 2 0.92742 0.92974 0.14689 0.14739 0.71855 0.71839 0.51929 0.51828 150 150 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk bulk bulk   China 2022-10-19 Adult Adult Eye Sensory System
71706 71706 SRR21966815 SRX17950294 SRS15467722 SRP403321 PRJNA891767 RNA seq of retina of zebrafish PRJNA891767 Other We performed bulk RNA sequencing on the zebrafish in an aim to identify the differential expression genes between retinal inflammation and normal group.       saline 72hpi 1 saline 72hpi 1   isolate:not applicable|age:6 month|sex:not applicable|tissue:retina|replicate:saline 72hpi biological replicate1|BioSampleModel:Model organism or animal                 RNA seq of retina saline 72hpi 1 saline 72hpi 1 RNA Extraction library construction and sequencingTotal RNA was extracted using Trizol reagent kit Invitrogen Carlsbad CA USA according to the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer Agilent Technologies Palo Alto CA USA and checked using RNase free agarose gel electrophoresis. post total RNA was extracted eukaryotic mRNA was enriched by OligodT beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second strand cDNA were synthesized by DNA polymerase I RNase H dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kitQiagen Venlo The Netherlands end repaired polyA added and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis PCR amplified and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. Guangzhou China.     RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP403321     saline_72hpi_1_1.fq.gz saline_72hpi_1_2.fq.gz fastq fastq 6779066400.0 22596888.0 saline 72hpi 1 1.fq.gz 0:150 1:150 A:1892919290;C:1494086551;G:1508786401;T:1883056956;N:217202 150 150     1892919290 1494086551 1508786401 1883056956 217202 SRX17950294 SRS15467722 SRA1523113 The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital|Henan International Joint Research Laboratory for The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital 2 0.92935 0.93091 0.14851 0.14735 0.71547 0.71315 0.5153 0.51561 150 150 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk bulk bulk   China 2022-10-19 Adult Adult Eye Sensory System
71707 71707 SRR21966816 SRX17950293 SRS15467721 SRP403321 PRJNA891767 RNA seq of retina of zebrafish PRJNA891767 Other We performed bulk RNA sequencing on the zebrafish in an aim to identify the differential expression genes between retinal inflammation and normal group.       saline 24hpi 3 saline 24hpi 3   isolate:not applicable|age:6 month|sex:not applicable|tissue:retina|replicate:saline 24hpi biological replicate3|BioSampleModel:Model organism or animal                 RNA seq of retina saline 24hpi 3 saline 24hpi 3 RNA Extraction library construction and sequencingTotal RNA was extracted using Trizol reagent kit Invitrogen Carlsbad CA USA according to the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer Agilent Technologies Palo Alto CA USA and checked using RNase free agarose gel electrophoresis. post total RNA was extracted eukaryotic mRNA was enriched by OligodT beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second strand cDNA were synthesized by DNA polymerase I RNase H dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kitQiagen Venlo The Netherlands end repaired polyA added and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis PCR amplified and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. Guangzhou China.     RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP403321     saline_24hpi_3_1.fq.gz saline_24hpi_3_2.fq.gz fastq fastq 6649910100.0 22166367.0 saline 24hpi 3 1.fq.gz 0:150 1:150 A:1855648802;C:1467136253;G:1483529961;T:1843379854;N:215230 150 150     1855648802 1467136253 1483529961 1843379854 215230 SRX17950293 SRS15467721 SRA1523113 The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital|Henan International Joint Research Laboratory for The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital 2 0.92959 0.93164 0.14859 0.14863 0.71287 0.71155 0.50104 0.50312 150 150 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk bulk bulk   China 2022-10-19 Adult Adult Eye Sensory System
71708 71708 SRR21966817 SRX17950292 SRS15467720 SRP403321 PRJNA891767 RNA seq of retina of zebrafish PRJNA891767 Other We performed bulk RNA sequencing on the zebrafish in an aim to identify the differential expression genes between retinal inflammation and normal group.       LPS 4hpi 3 LPS 4hpi 3   isolate:not applicable|age:6 month|sex:not applicable|tissue:retina|replicate:LPS 4hpi biological replicate3|BioSampleModel:Model organism or animal                 RNA seq of retina LPS 4hpi 3 LPS 4hpi 3 RNA Extraction library construction and sequencingTotal RNA was extracted using Trizol reagent kit Invitrogen Carlsbad CA USA according to the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer Agilent Technologies Palo Alto CA USA and checked using RNase free agarose gel electrophoresis. post total RNA was extracted eukaryotic mRNA was enriched by OligodT beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second strand cDNA were synthesized by DNA polymerase I RNase H dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kitQiagen Venlo The Netherlands end repaired polyA added and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis PCR amplified and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. Guangzhou China.     RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP403321     LPS_4hpi_3_1.fq.gz LPS_4hpi_3_2.fq.gz fastq fastq 5464551600.0 18215172.0 LPS 4hpi 3 1.fq.gz 0:150 1:150 A:1533153032;C:1197563026;G:1209118857;T:1524562004;N:154681 150 150     1533153032 1197563026 1209118857 1524562004 154681 SRX17950292 SRS15467720 SRA1523113 The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital|Henan International Joint Research Laboratory for The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital 2 0.93407 0.93358 0.14155 0.141 0.71855 0.71794 0.52207 0.5216 150 150 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk bulk bulk   China 2022-10-19 Adult Adult Eye Sensory System
71709 71709 SRR21966818 SRX17950291 SRS15467719 SRP403321 PRJNA891767 RNA seq of retina of zebrafish PRJNA891767 Other We performed bulk RNA sequencing on the zebrafish in an aim to identify the differential expression genes between retinal inflammation and normal group.       saline 24hpi 2 saline 24hpi 2   isolate:not applicable|age:6 month|sex:not applicable|tissue:retina|replicate:saline 24hpi biological replicate2|BioSampleModel:Model organism or animal                 RNA seq of retina saline 24hpi 2 saline 24hpi 2 RNA Extraction library construction and sequencingTotal RNA was extracted using Trizol reagent kit Invitrogen Carlsbad CA USA according to the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer Agilent Technologies Palo Alto CA USA and checked using RNase free agarose gel electrophoresis. post total RNA was extracted eukaryotic mRNA was enriched by OligodT beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second strand cDNA were synthesized by DNA polymerase I RNase H dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kitQiagen Venlo The Netherlands end repaired polyA added and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis PCR amplified and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. Guangzhou China.     RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP403321     saline_24hpi_2_1.fq.gz saline_24hpi_2_2.fq.gz fastq fastq 7233145200.0 24110484.0 saline 24hpi 2 1.fq.gz 0:150 1:150 A:2023236615;C:1591294466;G:1606319665;T:2012062580;N:231874 150 150     2023236615 1591294466 1606319665 2012062580 231874 SRX17950291 SRS15467719 SRA1523113 The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital|Henan International Joint Research Laboratory for The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital 2 0.92953 0.93246 0.14708 0.14779 0.71699 0.71603 0.50923 0.50973 150 150 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk bulk bulk   China 2022-10-19 Adult Adult Eye Sensory System
71710 71710 SRR21966819 SRX17950290 SRS15467718 SRP403321 PRJNA891767 RNA seq of retina of zebrafish PRJNA891767 Other We performed bulk RNA sequencing on the zebrafish in an aim to identify the differential expression genes between retinal inflammation and normal group.       saline 24hpi 1 saline 24hpi 1   isolate:not applicable|age:6 month|sex:not applicable|tissue:retina|replicate:saline 24hpi biological replicate1|BioSampleModel:Model organism or animal                 RNA seq of retina saline 24hpi 1 saline 24hpi 1 RNA Extraction library construction and sequencingTotal RNA was extracted using Trizol reagent kit Invitrogen Carlsbad CA USA according to the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer Agilent Technologies Palo Alto CA USA and checked using RNase free agarose gel electrophoresis. post total RNA was extracted eukaryotic mRNA was enriched by OligodT beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second strand cDNA were synthesized by DNA polymerase I RNase H dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kitQiagen Venlo The Netherlands end repaired polyA added and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis PCR amplified and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. Guangzhou China.     RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP403321     saline_24hpi_1_1.fq.gz saline_24hpi_1_2.fq.gz fastq fastq 6078786300.0 20262621.0 saline 24hpi 1 1.fq.gz 0:150 1:150 A:1698044840;C:1340218683;G:1354629060;T:1685699494;N:194223 150 150     1698044840 1340218683 1354629060 1685699494 194223 SRX17950290 SRS15467718 SRA1523113 The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital|Henan International Joint Research Laboratory for The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital 2 0.93162 0.93365 0.14384 0.144 0.71936 0.71871 0.50482 0.50161 150 150 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk bulk bulk   China 2022-10-19 Adult Adult Eye Sensory System
71711 71711 SRR21966820 SRX17950289 SRS15467717 SRP403321 PRJNA891767 RNA seq of retina of zebrafish PRJNA891767 Other We performed bulk RNA sequencing on the zebrafish in an aim to identify the differential expression genes between retinal inflammation and normal group.       saline 12hpi 3 saline 12hpi 3   isolate:not applicable|age:6 month|sex:not applicable|tissue:retina|replicate:saline 12hpi biological replicate3|BioSampleModel:Model organism or animal                 RNA seq of retina saline 12hpi 3 saline 12hpi 3 RNA Extraction library construction and sequencingTotal RNA was extracted using Trizol reagent kit Invitrogen Carlsbad CA USA according to the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer Agilent Technologies Palo Alto CA USA and checked using RNase free agarose gel electrophoresis. post total RNA was extracted eukaryotic mRNA was enriched by OligodT beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second strand cDNA were synthesized by DNA polymerase I RNase H dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kitQiagen Venlo The Netherlands end repaired polyA added and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis PCR amplified and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. Guangzhou China.     RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP403321     saline_12hpi_3_1.fq.gz saline_12hpi_3_2.fq.gz fastq fastq 5472245100.0 18240817.0 saline 12hpi 3 1.fq.gz 0:150 1:150 A:1521796644;C:1211080389;G:1225571730;T:1513617312;N:179025 150 150     1521796644 1211080389 1225571730 1513617312 179025 SRX17950289 SRS15467717 SRA1523113 The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital|Henan International Joint Research Laboratory for The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital 2 0.93247 0.93399 0.13756 0.13795 0.71877 0.71883 0.4937 0.48837 150 150 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk bulk bulk   China 2022-10-19 Adult Adult Eye Sensory System
71712 71712 SRR21966821 SRX17950288 SRS15467716 SRP403321 PRJNA891767 RNA seq of retina of zebrafish PRJNA891767 Other We performed bulk RNA sequencing on the zebrafish in an aim to identify the differential expression genes between retinal inflammation and normal group.       saline 12hpi 2 saline 12hpi 2   isolate:not applicable|age:6 month|sex:not applicable|tissue:retina|replicate:saline 12hpi biological replicate2|BioSampleModel:Model organism or animal                 RNA seq of retina saline 12hpi 2 saline 12hpi 2 RNA Extraction library construction and sequencingTotal RNA was extracted using Trizol reagent kit Invitrogen Carlsbad CA USA according to the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer Agilent Technologies Palo Alto CA USA and checked using RNase free agarose gel electrophoresis. post total RNA was extracted eukaryotic mRNA was enriched by OligodT beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second strand cDNA were synthesized by DNA polymerase I RNase H dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kitQiagen Venlo The Netherlands end repaired polyA added and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis PCR amplified and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. Guangzhou China.     RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP403321     saline_12hpi_2_1.fq.gz saline_12hpi_2_2.fq.gz fastq fastq 5885477400.0 19618258.0 saline 12hpi 2 1.fq.gz 0:150 1:150 A:1642325992;C:1298124930;G:1313140275;T:1631695965;N:190238 150 150     1642325992 1298124930 1313140275 1631695965 190238 SRX17950288 SRS15467716 SRA1523113 The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital|Henan International Joint Research Laboratory for The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital 2 0.93111 0.93297 0.14002 0.14078 0.71642 0.71559 0.49307 0.49089 150 150 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk bulk bulk   China 2022-10-19 Adult Adult Eye Sensory System
71713 71713 SRR21966822 SRX17950287 SRS15467715 SRP403321 PRJNA891767 RNA seq of retina of zebrafish PRJNA891767 Other We performed bulk RNA sequencing on the zebrafish in an aim to identify the differential expression genes between retinal inflammation and normal group.       saline 12hpi 1 saline 12hpi 1   isolate:not applicable|age:6 month|sex:not applicable|tissue:retina|replicate:saline 12hpi biological replicate1|BioSampleModel:Model organism or animal                 RNA seq of retina saline 12hpi 1 saline 12hpi 1 RNA Extraction library construction and sequencingTotal RNA was extracted using Trizol reagent kit Invitrogen Carlsbad CA USA according to the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer Agilent Technologies Palo Alto CA USA and checked using RNase free agarose gel electrophoresis. post total RNA was extracted eukaryotic mRNA was enriched by OligodT beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second strand cDNA were synthesized by DNA polymerase I RNase H dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kitQiagen Venlo The Netherlands end repaired polyA added and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis PCR amplified and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. Guangzhou China.     RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP403321     saline_12hpi_1_1.fq.gz saline_12hpi_1_2.fq.gz fastq fastq 13424687400.0 44748958.0 saline 12hpi 1 1.fq.gz 0:150 1:150 A:3721315477;C:2980358472;G:3018026284;T:3704553827;N:433340 150 150     3721315477 2980358472 3018026284 3704553827 433340 SRX17950287 SRS15467715 SRA1523113 The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital|Henan International Joint Research Laboratory for The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital 2 0.93225 0.93342 0.13753 0.13795 0.70806 0.70668 0.49478 0.49461 150 150 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk bulk bulk   China 2022-10-19 Adult Adult Eye Sensory System
71714 71714 SRR21966823 SRX17950286 SRS15467714 SRP403321 PRJNA891767 RNA seq of retina of zebrafish PRJNA891767 Other We performed bulk RNA sequencing on the zebrafish in an aim to identify the differential expression genes between retinal inflammation and normal group.       saline 4hpi 3 saline 4hpi 3   isolate:not applicable|age:6 month|sex:not applicable|tissue:retina|replicate:saline 4hpi biological replicate3|BioSampleModel:Model organism or animal                 RNA seq of retina saline 4hpi 3 saline 4hpi 3 RNA Extraction library construction and sequencingTotal RNA was extracted using Trizol reagent kit Invitrogen Carlsbad CA USA according to the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer Agilent Technologies Palo Alto CA USA and checked using RNase free agarose gel electrophoresis. post total RNA was extracted eukaryotic mRNA was enriched by OligodT beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second strand cDNA were synthesized by DNA polymerase I RNase H dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kitQiagen Venlo The Netherlands end repaired polyA added and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis PCR amplified and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. Guangzhou China.     RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP403321     saline_4hpi_3_1.fq.gz saline_4hpi_3_2.fq.gz fastq fastq 6794057100.0 22646857.0 saline 4hpi 3 1.fq.gz 0:150 1:150 A:1888459583;C:1506738618;G:1522940393;T:1875697125;N:221381 150 150     1888459583 1506738618 1522940393 1875697125 221381 SRX17950286 SRS15467714 SRA1523113 The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital|Henan International Joint Research Laboratory for The First Affiliated Hospital of Zhengzhou University, Henan Province Eye Hospital 2 0.93375 0.9351 0.13089 0.13151 0.71938 0.71954 0.51685 0.51377 150 150 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk bulk bulk   China 2022-10-19 Adult Adult Eye Sensory System

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CREATE TABLE run_metadata("run.accession" VARCHAR, "experiment.accession" VARCHAR, "sample.accession" VARCHAR, "study.accession" VARCHAR, bioproject VARCHAR, "study.title" VARCHAR, "study.alias" VARCHAR, "study.type" VARCHAR, "study.abstract" VARCHAR, "study.attributes" VARCHAR, "study.PMIDs" VARCHAR, "sample.description" VARCHAR, "sample.title" VARCHAR, "sample.alias" VARCHAR, "sample.centername" VARCHAR, "sample.attributes" VARCHAR, "GEOsample.title" VARCHAR, "GEOsample.dataprocessing" VARCHAR, "GEOsample.source" VARCHAR, "GEOsample.treatmentprotocol" VARCHAR, "GEOsample.extractprotocol" VARCHAR, "GEOsample.growthprotocol" VARCHAR, "GEOsample.characteristics" VARCHAR, "GEOsample.accession" VARCHAR, "experiment.title" VARCHAR, "experiment.alias" VARCHAR, "experiment.library_name" VARCHAR, "experiment.design_description" VARCHAR, "experiment.library_construction_protocol" VARCHAR, "experiment.attributes" VARCHAR, "experiment.library_strategy" VARCHAR, "experiment.library_source" VARCHAR, "experiment.library_selection" VARCHAR, "experiment.library_layout" VARCHAR, "experiment.platform" VARCHAR, "experiment.instrument_model" VARCHAR, "experiment.spot_descriptor" VARCHAR, "experiment.study_ref" VARCHAR, "run.title" VARCHAR, "run.attributes" VARCHAR, "run.filename" VARCHAR, "run.semantic_name" VARCHAR, "run.total_bases" DOUBLE, "run.total_spots" DOUBLE, "run.alias" VARCHAR, "run.read_lengths" VARCHAR, "run.base_counts" VARCHAR, "run.r1_length" BIGINT, "run.r2_length" BIGINT, "run.r3_length" BIGINT, "run.r4_length" BIGINT, "run.Acount" BIGINT, "run.Ccount" BIGINT, "run.Gcount" BIGINT, "run.Tcount" BIGINT, "run.Ncount" BIGINT, "run.experiment" VARCHAR, "run.pool_member" VARCHAR, "submission.accession" VARCHAR, "submission.srasource" VARCHAR, "submission.bioprojectsource" VARCHAR, "seqdetective.n_mates" BIGINT, "seqdetective.mapping_rate.mate1" DOUBLE, "seqdetective.mapping_rate.mate2" DOUBLE, "seqdetective.nofeature_rate.mate1" DOUBLE, "seqdetective.nofeature_rate.mate2" DOUBLE, "seqdetective.sparsity.mate1" DOUBLE, "seqdetective.sparsity.mate2" DOUBLE, "seqdetective.pos_strand_rate.mate1" DOUBLE, "seqdetective.pos_strand_rate.mate2" DOUBLE, "seqdetective.readlen.mate1" BIGINT, "seqdetective.readlen.mate2" BIGINT, "seqdetective.judgement.mate1" VARCHAR, "seqdetective.judgement.mate2" VARCHAR, "seqdetective.judgement.reason" VARCHAR, platform_family VARCHAR, instrument_generation VARCHAR, read_bias VARCHAR, selection_class VARCHAR, prep_kit VARCHAR, sc_or_bulk VARCHAR, tech_class VARCHAR, technology VARCHAR, tech_variant VARCHAR, "submission.bioprojectsource.country" VARCHAR, earliest_date DATE, devstage_curation VARCHAR, devstage_curation_coarse VARCHAR, tissue_curation VARCHAR, tissue_curation_coarse VARCHAR);;
CREATE INDEX idx_run_bioproject ON run_metadata(bioproject);;
CREATE INDEX idx_run_run_accession ON run_metadata("run.accession");;