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440 rows where devstage_curation = "Undetermined" and tissue_curation = "Eye"

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technology 5

devstage_curation_coarse 5

experiment.library_selection 3

experiment.platform 2

experiment.library_strategy 2

experiment.library_source 2

experiment.library_layout 2

tissue_curation_coarse 1

tissue_curation 1

  • Eye · 440 ✖

devstage_curation 1

  • Undetermined · 440 ✖
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
2319 2319 ERR1289947 ERX1361553 ERS954843 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647694 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:23:00Z|External Id:SAMEA3647694|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:23:00Z|INSDC status:public|Submitter Id:ATH5 2 sc 2454965|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:ATH5 2 sc 2454965|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 2#12 15249493 Illumina sequencing of library 15249493 constructed from sample accession ERS954843 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 2. This submission includes reads tagged with the sequence TAGGCATGAGAGTAGA. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_2#12.cram cram 2492206600.0 12461033.0 SC RUN 18222 2#12 0:100 1:100 A:661236051;C:588399772;G:571478215;T:671080581;N:11981 100 100     661236051 588399772 571478215 671080581 11981 ERX1361553 ERS954843 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.94054 0.94077 0.10025 0.10255 0.75779 0.76019 0.4807 0.47966 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2320 2320 ERR1289946 ERX1361552 ERS954842 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647693 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:23:00Z|External Id:SAMEA3647693|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:23:00Z|INSDC status:public|Submitter Id:WT ctrl sc 2454964|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:WT ctrl sc 2454964|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 2#11 15249492 Illumina sequencing of library 15249492 constructed from sample accession ERS954842 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 2. This submission includes reads tagged with the sequence TAGGCATGTATCCTCT. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_2#11.cram cram 2748114600.0 13740573.0 SC RUN 18222 2#11 0:100 1:100 A:747132237;C:630918171;G:613637334;T:756413030;N:13828 100 100     747132237 630918171 613637334 756413030 13828 ERX1361552 ERS954842 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.91201 0.911 0.09071 0.09156 0.88994 0.88988 0.47118 0.49948 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2321 2321 ERR1289945 ERX1361551 ERS954841 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647692 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:22:59Z|External Id:SAMEA3647692|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:22:59Z|INSDC status:public|Submitter Id:RX2 ATH5 GFP3 sc 2454963|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:RX2 ATH5 GFP3 sc 2454963|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 2#10 15249491 Illumina sequencing of library 15249491 constructed from sample accession ERS954841 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 2. This submission includes reads tagged with the sequence TAGGCATGCTCTCTAT. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_2#10.cram cram 3366968000.0 16834840.0 SC RUN 18222 2#10 0:100 1:100 A:881918684;C:806052916;G:790157920;T:888821188;N:17292 100 100     881918684 806052916 790157920 888821188 17292 ERX1361551 ERS954841 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.94171 0.94098 0.0849 0.08607 0.74862 0.75024 0.50963 0.50955 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2322 2322 ERR1289944 ERX1361550 ERS954840 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647691 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:25:54Z|External Id:SAMEA3647691|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:25:54Z|INSDC status:public|Submitter Id:RX2 ATH5 GFP2 sc 2454962|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:RX2 ATH5 GFP2 sc 2454962|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 2#9 15249490 Illumina sequencing of library 15249490 constructed from sample accession ERS954840 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 2. This submission includes reads tagged with the sequence TAGGCATGTAGATCGC. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_2#9.cram cram 3222261000.0 16111305.0 SC RUN 18222 2#9 0:100 1:100 A:819997366;C:795302699;G:777099203;T:829845734;N:15998 100 100     819997366 795302699 777099203 829845734 15998 ERX1361550 ERS954840 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.94529 0.94495 0.0603 0.06143 0.76157 0.76238 0.48847 0.4882 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2323 2323 ERR1289943 ERX1361549 ERS954839 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647690 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:22:57Z|External Id:SAMEA3647690|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:22:57Z|INSDC status:public|Submitter Id:RX2 GFP2 high sc 2454961|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:RX2 GFP2 high sc 2454961|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 2#8 15249489 Illumina sequencing of library 15249489 constructed from sample accession ERS954839 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 2. This submission includes reads tagged with the sequence GGACTCCTCTAAGCCT. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_2#8.cram cram 3834749800.0 19173749.0 SC RUN 18222 2#8 0:100 1:100 A:992673645;C:929008642;G:906766397;T:1006281701;N:19415 100 100     992673645 929008642 906766397 1006281701 19415 ERX1361549 ERS954839 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.94351 0.94297 0.08055 0.08157 0.72853 0.73095 0.52148 0.51755 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2324 2324 ERR1289942 ERX1361548 ERS954838 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647689 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:22:57Z|External Id:SAMEA3647689|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:22:57Z|INSDC status:public|Submitter Id:RX2 GFP2 low sc 2454960|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:RX2 GFP2 low sc 2454960|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 2#7 15249488 Illumina sequencing of library 15249488 constructed from sample accession ERS954838 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 2. This submission includes reads tagged with the sequence GGACTCCTAAGGAGTA. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_2#7.cram cram 1076581000.0 5382905.0 SC RUN 18222 2#7 0:100 1:100 A:285946062;C:254142940;G:242866633;T:293619980;N:5385 100 100     285946062 254142940 242866633 293619980 5385 ERX1361548 ERS954838 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.89818 0.89819 0.1022 0.10292 0.89919 0.89852 0.51448 0.51223 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2325 2325 ERR1289941 ERX1361547 ERS954837 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647688 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:25:54Z|External Id:SAMEA3647688|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:25:54Z|INSDC status:public|Submitter Id:GFAP ATH5 GFP2 sc 2454959|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:GFAP ATH5 GFP2 sc 2454959|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 2#6 15249487 Illumina sequencing of library 15249487 constructed from sample accession ERS954837 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 2. This submission includes reads tagged with the sequence GGACTCCTACTGCATA. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_2#6.cram cram 3172834800.0 15864174.0 SC RUN 18222 2#6 0:100 1:100 A:841262183;C:748974758;G:730474017;T:852107947;N:15895 100 100     841262183 748974758 730474017 852107947 15895 ERX1361547 ERS954837 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.93515 0.93517 0.10079 0.10267 0.73403 0.73511 0.49831 0.47169 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2326 2326 ERR1289940 ERX1361546 ERS954836 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647687 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:25:54Z|External Id:SAMEA3647687|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:25:54Z|INSDC status:public|Submitter Id:ATH5 1 sc 2454958|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:ATH5 1 sc 2454958|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 2#5 15249486 Illumina sequencing of library 15249486 constructed from sample accession ERS954836 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 2. This submission includes reads tagged with the sequence GGACTCCTGTAAGGAG. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_2#5.cram cram 1188728400.0 5943642.0 SC RUN 18222 2#5 0:100 1:100 A:322290258;C:273659598;G:266021599;T:326751009;N:5936 100 100     322290258 273659598 266021599 326751009 5936 ERX1361546 ERS954836 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.92438 0.92391 0.12338 0.1251 0.76288 0.76479 0.48734 0.47777 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2327 2327 ERR1289939 ERX1361545 ERS954835 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647686 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:25:54Z|External Id:SAMEA3647686|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:25:54Z|INSDC status:public|Submitter Id:GFAP ATH5 GFP1 sc 2454957|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:GFAP ATH5 GFP1 sc 2454957|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 2#4 15249485 Illumina sequencing of library 15249485 constructed from sample accession ERS954835 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 2. This submission includes reads tagged with the sequence GGACTCCTAGAGTAGA. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_2#4.cram cram 2335470400.0 11677352.0 SC RUN 18222 2#4 0:100 1:100 A:611730664;C:560095252;G:541373953;T:622259122;N:11409 100 100     611730664 560095252 541373953 622259122 11409 ERX1361545 ERS954835 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.93481 0.93465 0.10573 0.10811 0.77135 0.77303 0.50016 0.50137 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2328 2328 ERR1289938 ERX1361544 ERS954834 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647685 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:22:57Z|External Id:SAMEA3647685|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:22:57Z|INSDC status:public|Submitter Id:RX2 ATH5 GFP1 sc 2454956|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:RX2 ATH5 GFP1 sc 2454956|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 2#3 15249484 Illumina sequencing of library 15249484 constructed from sample accession ERS954834 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 2. This submission includes reads tagged with the sequence GGACTCCTTATCCTCT. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_2#3.cram cram 2932765000.0 14663825.0 SC RUN 18222 2#3 0:100 1:100 A:762543647;C:708033363;G:692599413;T:769573475;N:15102 100 100     762543647 708033363 692599413 769573475 15102 ERX1361544 ERS954834 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.94442 0.94374 0.07322 0.07439 0.74383 0.74531 0.50604 0.51608 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2329 2329 ERR1289937 ERX1361543 ERS954833 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647684 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:25:15Z|External Id:SAMEA3647684|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:25:15Z|INSDC status:public|Submitter Id:RX2 GFP1 sc 2454955|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:RX2 GFP1 sc 2454955|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 2#2 15249483 Illumina sequencing of library 15249483 constructed from sample accession ERS954833 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 2. This submission includes reads tagged with the sequence GGACTCCTCTCTCTAT. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_2#2.cram cram 2432398000.0 12161990.0 SC RUN 18222 2#2 0:100 1:100 A:624543123;C:594774023;G:581130641;T:631937939;N:12274 100 100     624543123 594774023 581130641 631937939 12274 ERX1361543 ERS954833 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.94225 0.94103 0.08221 0.0823 0.79289 0.79383 0.51106 0.5129 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2330 2330 ERR1289936 ERX1361542 ERS954832 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647683 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:22:57Z|External Id:SAMEA3647683|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:22:57Z|INSDC status:public|Submitter Id:RX2 ctrl noGFP sc 2454954|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:RX2 ctrl noGFP sc 2454954|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 2#1 15249482 Illumina sequencing of library 15249482 constructed from sample accession ERS954832 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 2. This submission includes reads tagged with the sequence GGACTCCTTAGATCGC. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_2#1.cram cram 361900800.0 1809504.0 SC RUN 18222 2#1 0:100 1:100 A:84553945;C:97259075;G:92615955;T:87470072;N:1753 100 100     84553945 97259075 92615955 87470072 1753 ERX1361542 ERS954832 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.94897 0.95014 0.07743 0.07772 0.71969 0.72058 0.43784 0.44067 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2331 2331 ERR1289935 ERX1361541 ERS954843 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647694 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:23:00Z|External Id:SAMEA3647694|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:23:00Z|INSDC status:public|Submitter Id:ATH5 2 sc 2454965|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:ATH5 2 sc 2454965|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 1#12 15249493 Illumina sequencing of library 15249493 constructed from sample accession ERS954843 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 1. This submission includes reads tagged with the sequence TAGGCATGAGAGTAGA. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_1#12.cram cram 2487554200.0 12437771.0 SC RUN 18222 1#12 0:100 1:100 A:660028585;C:587257902;G:570415230;T:669842822;N:9661 100 100     660028585 587257902 570415230 669842822 9661 ERX1361541 ERS954843 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.94059 0.9397 0.09914 0.1011 0.75597 0.75862 0.48558 0.48548 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2332 2332 ERR1289934 ERX1361540 ERS954842 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647693 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:23:00Z|External Id:SAMEA3647693|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:23:00Z|INSDC status:public|Submitter Id:WT ctrl sc 2454964|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:WT ctrl sc 2454964|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 1#11 15249492 Illumina sequencing of library 15249492 constructed from sample accession ERS954842 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 1. This submission includes reads tagged with the sequence TAGGCATGTATCCTCT. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_1#11.cram cram 2740851200.0 13704256.0 SC RUN 18222 1#11 0:100 1:100 A:745054545;C:629082779;G:612051041;T:754652050;N:10785 100 100     745054545 629082779 612051041 754652050 10785 ERX1361540 ERS954842 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.91349 0.91229 0.09216 0.09351 0.88962 0.89043 0.48978 0.5028 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2333 2333 ERR1289933 ERX1361539 ERS954841 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647692 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:22:59Z|External Id:SAMEA3647692|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:22:59Z|INSDC status:public|Submitter Id:RX2 ATH5 GFP3 sc 2454963|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:RX2 ATH5 GFP3 sc 2454963|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 1#10 15249491 Illumina sequencing of library 15249491 constructed from sample accession ERS954841 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 1. This submission includes reads tagged with the sequence TAGGCATGCTCTCTAT. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_1#10.cram cram 3357950400.0 16789752.0 SC RUN 18222 1#10 0:100 1:100 A:879637755;C:803810252;G:787961249;T:886528291;N:12853 100 100     879637755 803810252 787961249 886528291 12853 ERX1361539 ERS954841 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.94064 0.94017 0.08377 0.08517 0.74909 0.75136 0.50723 0.50903 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2334 2334 ERR1289932 ERX1361538 ERS954840 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647691 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:25:54Z|External Id:SAMEA3647691|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:25:54Z|INSDC status:public|Submitter Id:RX2 ATH5 GFP2 sc 2454962|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:RX2 ATH5 GFP2 sc 2454962|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 1#9 15249490 Illumina sequencing of library 15249490 constructed from sample accession ERS954840 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 1. This submission includes reads tagged with the sequence TAGGCATGTAGATCGC. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_1#9.cram cram 3206581200.0 16032906.0 SC RUN 18222 1#9 0:100 1:100 A:816111545;C:791236086;G:773298814;T:825922398;N:12357 100 100     816111545 791236086 773298814 825922398 12357 ERX1361538 ERS954840 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.94549 0.94564 0.06037 0.06105 0.76043 0.76171 0.48313 0.49555 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2335 2335 ERR1289931 ERX1361537 ERS954839 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647690 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:22:57Z|External Id:SAMEA3647690|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:22:57Z|INSDC status:public|Submitter Id:RX2 GFP2 high sc 2454961|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:RX2 GFP2 high sc 2454961|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 1#8 15249489 Illumina sequencing of library 15249489 constructed from sample accession ERS954839 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 1. This submission includes reads tagged with the sequence GGACTCCTCTAAGCCT. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_1#8.cram cram 3822332200.0 19111661.0 SC RUN 18222 1#8 0:100 1:100 A:989542225;C:925823012;G:903835738;T:1003115980;N:15245 100 100     989542225 925823012 903835738 1003115980 15245 ERX1361537 ERS954839 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.94289 0.94174 0.07966 0.08004 0.72764 0.72906 0.51976 0.52464 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2336 2336 ERR1289930 ERX1361536 ERS954838 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647689 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:22:57Z|External Id:SAMEA3647689|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:22:57Z|INSDC status:public|Submitter Id:RX2 GFP2 low sc 2454960|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:RX2 GFP2 low sc 2454960|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 1#7 15249488 Illumina sequencing of library 15249488 constructed from sample accession ERS954838 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 1. This submission includes reads tagged with the sequence GGACTCCTAAGGAGTA. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_1#7.cram cram 1072510200.0 5362551.0 SC RUN 18222 1#7 0:100 1:100 A:284863902;C:253046005;G:241968135;T:292627849;N:4309 100 100     284863902 253046005 241968135 292627849 4309 ERX1361536 ERS954838 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.89703 0.89873 0.10191 0.10317 0.89921 0.8984 0.50884 0.51319 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2337 2337 ERR1289929 ERX1361535 ERS954837 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647688 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:25:54Z|External Id:SAMEA3647688|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:25:54Z|INSDC status:public|Submitter Id:GFAP ATH5 GFP2 sc 2454959|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:GFAP ATH5 GFP2 sc 2454959|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 1#6 15249487 Illumina sequencing of library 15249487 constructed from sample accession ERS954837 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 1. This submission includes reads tagged with the sequence GGACTCCTACTGCATA. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_1#6.cram cram 3159715800.0 15798579.0 SC RUN 18222 1#6 0:100 1:100 A:837700548;C:745877706;G:727508293;T:848616717;N:12536 100 100     837700548 745877706 727508293 848616717 12536 ERX1361535 ERS954837 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.93397 0.93362 0.10011 0.10205 0.73474 0.73612 0.49003 0.49085 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2338 2338 ERR1289928 ERX1361534 ERS954836 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647687 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:25:54Z|External Id:SAMEA3647687|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:25:54Z|INSDC status:public|Submitter Id:ATH5 1 sc 2454958|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:ATH5 1 sc 2454958|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 1#5 15249486 Illumina sequencing of library 15249486 constructed from sample accession ERS954836 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 1. This submission includes reads tagged with the sequence GGACTCCTGTAAGGAG. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_1#5.cram cram 1184332600.0 5921663.0 SC RUN 18222 1#5 0:100 1:100 A:321129432;C:272595945;G:264945795;T:325656767;N:4661 100 100     321129432 272595945 264945795 325656767 4661 ERX1361534 ERS954836 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.92372 0.92353 0.12445 0.12616 0.76339 0.76495 0.48995 0.48259 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2339 2339 ERR1289927 ERX1361533 ERS954835 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647686 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:25:54Z|External Id:SAMEA3647686|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:25:54Z|INSDC status:public|Submitter Id:GFAP ATH5 GFP1 sc 2454957|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:GFAP ATH5 GFP1 sc 2454957|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 1#4 15249485 Illumina sequencing of library 15249485 constructed from sample accession ERS954835 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 1. This submission includes reads tagged with the sequence GGACTCCTAGAGTAGA. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_1#4.cram cram 2331489600.0 11657448.0 SC RUN 18222 1#4 0:100 1:100 A:610715735;C:559128165;G:540406653;T:621229940;N:9107 100 100     610715735 559128165 540406653 621229940 9107 ERX1361533 ERS954835 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.9338 0.93348 0.10518 0.10783 0.77155 0.77374 0.49506 0.49808 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2340 2340 ERR1289926 ERX1361532 ERS954834 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647685 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:22:57Z|External Id:SAMEA3647685|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:22:57Z|INSDC status:public|Submitter Id:RX2 ATH5 GFP1 sc 2454956|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:RX2 ATH5 GFP1 sc 2454956|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 1#3 15249484 Illumina sequencing of library 15249484 constructed from sample accession ERS954834 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 1. This submission includes reads tagged with the sequence GGACTCCTTATCCTCT. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_1#3.cram cram 2923134800.0 14615674.0 SC RUN 18222 1#3 0:100 1:100 A:760057089;C:705694497;G:690249046;T:767122385;N:11783 100 100     760057089 705694497 690249046 767122385 11783 ERX1361532 ERS954834 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.94509 0.94377 0.07431 0.07512 0.74367 0.74525 0.50981 0.51595 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2341 2341 ERR1289925 ERX1361531 ERS954833 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647684 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:25:15Z|External Id:SAMEA3647684|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:25:15Z|INSDC status:public|Submitter Id:RX2 GFP1 sc 2454955|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:RX2 GFP1 sc 2454955|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 1#2 15249483 Illumina sequencing of library 15249483 constructed from sample accession ERS954833 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 1. This submission includes reads tagged with the sequence GGACTCCTCTCTCTAT. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_1#2.cram cram 2423614200.0 12118071.0 SC RUN 18222 1#2 0:100 1:100 A:622254096;C:592596967;G:579007199;T:629746522;N:9416 100 100     622254096 592596967 579007199 629746522 9416 ERX1361531 ERS954833 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.94151 0.94151 0.08127 0.08228 0.79091 0.79235 0.51421 0.50953 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
2342 2342 ERR1289924 ERX1361530 ERS954832 ERP012920 PRJEB11523 Zebrafish eye populations transcriptomics Zebrafish_eye_populations_transcriptomics-sc-3967 Transcriptome Analysis To characterise the transcriptome of the stem cells and the proliferative progenitors in the retina of 5 dpf zebrafish Danio rerio eyes were prepared as follows: 20 30 eyes per condition were collected dissociated and FACS sorted. The samples were collected to RLT buffer and RNA extraction was immediately performed. cDNA and Nextera XT libraries were generated and subsequent sequencing was carried out using the Illumina HiSeq 2500 platform. ArrayExpress:E ERAD 441       SAMEA3647683 SC ArrayExpress OrganismPart:eye cell populations|ArrayExpress Sex:mixed|ArrayExpress Species:Danio rerio|ENA FIRST PUBLIC:2016 02 23T15:26:28Z|ENA LAST UPDATE:2018 03 09T09:22:57Z|External Id:SAMEA3647683|INSDC center name:SC|INSDC first public:2016 02 23T15:26:28Z|INSDC last update:2018 03 09T09:22:57Z|INSDC status:public|Submitter Id:RX2 ctrl noGFP sc 2454954|common name:zebrafish|sample description:eye cell populations from zebrafish embryo|sample name:RX2 ctrl noGFP sc 2454954|scientific name:Danio rerio                 Illumina HiSeq 2500 paired end sequencing SC EXP 18222 1#1 15249482 Illumina sequencing of library 15249482 constructed from sample accession ERS954832 for study accession ERP012920. This is part of an Illumina multiplexed sequencing run 18222 1. This submission includes reads tagged with the sequence GGACTCCTTAGATCGC. Nextera dual index qPCR only   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina HiSeq 2500   ERP012920 Illumina HiSeq 2500 paired end sequencing ENA FIRST PUBLIC:2016 02 23|ENA LAST UPDATE:2018 11 16 18222_1#1.cram cram 359779800.0 1798899.0 SC RUN 18222 1#1 0:100 1:100 A:84065568;C:96668644;G:92071125;T:86973168;N:1295 100 100     84065568 96668644 92071125 86973168 1295 ERX1361530 ERS954832 ERA565862 European Nucleotide Archive Wellcome Sanger Institute 2 0.9502 0.9506 0.07759 0.07791 0.71772 0.71827 0.44568 0.45005 100 100 B B biological fallback assumption illumina hiseq_era unknown cdna_unspecified nextera bulk unknown unknown   United Kingdom 2016-02-23 Undetermined Embryo Eye Sensory System
29092 29092 SRR27010839 SRX22703628 SRS19696103 SRP475450 PRJNA1047749 Differential expression in Muller glia of injured zebrafish in a mycb / context GSE249116 Transcriptome Analysis This experiment studies retinal regeneration using a zebrafish model with a focus on Muller glia reprogramming. Overall design: Three groups: controls Muller glia from uninjured retinas injured WT Muller glia from injured WT retina at 2 xxx post injury dpi and injured mycb / Muller glia from injured mycb / fish at 2dpi. parent bioproject:PRJNA1047494 pubmed:38984586   Muller glia from mycb / retinas 2 xxx post injury dpi rep2 [7131 ML 8] [7131 ML 8] GSM7926876   source name:mycb / Muller glia injured retina|tissue:mycb / Muller glia injured retina|genotype:mycb / |treatment:injured|geo loc name:missing|collection date:missing Muller glia from mycb / retinas 2 xxx post injury dpi rep2 [7131 ML 8] [7131 ML 8] University of Michigan Bioinformatics Core RNA seq pipeline "Watermelon" version 2.4.4 Sequence reads were trimmed for adaptor sequence/low quality sequence using Cutadapt v2.3. FastQC v0.11.8 was used to ensure the quality of data Trimmed sequence reads were mapped to GRCh38/hs19 using STAR v2.7.8a Quantitative count estimates to genes performed with RSEM v1.3.3 Assembly: GRCz11 Supplementary files format and content: RS11 deseq2 raw counts.txt Supplementary files format and content: tab delimited text with include expected counts values for each Sample mycb / Muller glia injured retina Needle poke injury to retina. Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   tissue:mycb / Muller glia injured retina|genotype:mycb / |treatment:injured GSM7926876 GSM7926876: Muller glia from mycb / retinas 2 xxx post injury dpi rep2 [7131 ML 8] [7131 ML 8]; Danio rerio; RNA Seq GSM7926876 r1 GSM7926876 1 Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP475450   loader:fastq load.py 7131-ML-8_GCTGGCCA-GCGTCCGT_S74_R1_001.fastq.gz 7131-ML-8_GCTGGCCA-GCGTCCGT_S74_R2_001.fastq.gz fastq fastq 27012387282.0 89444991.0 GSM7926876 r1 0:151 1:151 A:7324891140;C:5760958085;G:6796293318;T:7129968103;N:276636 151 151     7324891140 5760958085 6796293318 7129968103 276636 SRX22703628 SRS19696103 SRA1761650 University of Michigan University of Michigan 2 0.88985 0.86096 0.09217 0.08056 0.7473 0.7542 0.59862 0.5926 151 151 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk unknown unknown   United States 2023-12-01 Undetermined Undetermined Eye Sensory System
29093 29093 SRR27010840 SRX22703627 SRS19696102 SRP475450 PRJNA1047749 Differential expression in Muller glia of injured zebrafish in a mycb / context GSE249116 Transcriptome Analysis This experiment studies retinal regeneration using a zebrafish model with a focus on Muller glia reprogramming. Overall design: Three groups: controls Muller glia from uninjured retinas injured WT Muller glia from injured WT retina at 2 xxx post injury dpi and injured mycb / Muller glia from injured mycb / fish at 2dpi. parent bioproject:PRJNA1047494 pubmed:38984586   Muller glia from mycb / retinas 2 xxx post injury dpi rep1 [7131 ML 7] [7131 ML 7] GSM7926875   source name:mycb / Muller glia injured retina|tissue:mycb / Muller glia injured retina|genotype:mycb / |treatment:injured|geo loc name:missing|collection date:missing Muller glia from mycb / retinas 2 xxx post injury dpi rep1 [7131 ML 7] [7131 ML 7] University of Michigan Bioinformatics Core RNA seq pipeline "Watermelon" version 2.4.4 Sequence reads were trimmed for adaptor sequence/low quality sequence using Cutadapt v2.3. FastQC v0.11.8 was used to ensure the quality of data Trimmed sequence reads were mapped to GRCh38/hs19 using STAR v2.7.8a Quantitative count estimates to genes performed with RSEM v1.3.3 Assembly: GRCz11 Supplementary files format and content: RS11 deseq2 raw counts.txt Supplementary files format and content: tab delimited text with include expected counts values for each Sample mycb / Muller glia injured retina Needle poke injury to retina. Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   tissue:mycb / Muller glia injured retina|genotype:mycb / |treatment:injured GSM7926875 GSM7926875: Muller glia from mycb / retinas 2 xxx post injury dpi rep1 [7131 ML 7] [7131 ML 7]; Danio rerio; RNA Seq GSM7926875 r1 GSM7926875 1 Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP475450   loader:fastq load.py 7131-ML-7_TTACGGAC-ATTGGCCA_S73_R1_001.fastq.gz 7131-ML-7_TTACGGAC-ATTGGCCA_S73_R2_001.fastq.gz fastq fastq 27084816546.0 89684823.0 GSM7926875 r1 0:151 1:151 A:7489987820;C:5729672678;G:6569226079;T:7295655071;N:274898 151 151     7489987820 5729672678 6569226079 7295655071 274898 SRX22703627 SRS19696102 SRA1761650 University of Michigan University of Michigan 2 0.8887 0.84473 0.12952 0.10995 0.73338 0.74211 0.54826 0.56906 151 151 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk unknown unknown   United States 2023-12-01 Undetermined Undetermined Eye Sensory System
29094 29094 SRR27010841 SRX22703626 SRS19696101 SRP475450 PRJNA1047749 Differential expression in Muller glia of injured zebrafish in a mycb / context GSE249116 Transcriptome Analysis This experiment studies retinal regeneration using a zebrafish model with a focus on Muller glia reprogramming. Overall design: Three groups: controls Muller glia from uninjured retinas injured WT Muller glia from injured WT retina at 2 xxx post injury dpi and injured mycb / Muller glia from injured mycb / fish at 2dpi. parent bioproject:PRJNA1047494 pubmed:38984586   Muller glia from WT retinas 2 xxx post injury dpi rep2 [7131 ML 5] [7131 ML 5] GSM7926874   source name:WT Muller glia injured retina|tissue:WT Muller glia injured retina|genotype:WT|treatment:n1|geo loc name:missing|collection date:missing Muller glia from WT retinas 2 xxx post injury dpi rep2 [7131 ML 5] [7131 ML 5] University of Michigan Bioinformatics Core RNA seq pipeline "Watermelon" version 2.4.4 Sequence reads were trimmed for adaptor sequence/low quality sequence using Cutadapt v2.3. FastQC v0.11.8 was used to ensure the quality of data Trimmed sequence reads were mapped to GRCh38/hs19 using STAR v2.7.8a Quantitative count estimates to genes performed with RSEM v1.3.3 Assembly: GRCz11 Supplementary files format and content: RS11 deseq2 raw counts.txt Supplementary files format and content: tab delimited text with include expected counts values for each Sample WT Muller glia injured retina Needle poke injury to retina. Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   tissue:WT Muller glia injured retina|genotype:WT|treatment:n1 GSM7926874 GSM7926874: Muller glia from WT retinas 2 xxx post injury dpi rep2 [7131 ML 5] [7131 ML 5]; Danio rerio; RNA Seq GSM7926874 r1 GSM7926874 1 Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP475450   loader:fastq load.py 7131-ML-5_CAACCATA-ACCGGTTA_S71_R1_001.fastq.gz 7131-ML-5_CAACCATA-ACCGGTTA_S71_R2_001.fastq.gz fastq fastq 29094896836.0 96340718.0 GSM7926874 r1 0:151 1:151 A:7739727136;C:6356741244;G:7473778902;T:7524355633;N:293921 151 151     7739727136 6356741244 7473778902 7524355633 293921 SRX22703626 SRS19696101 SRA1761650 University of Michigan University of Michigan 2 0.88966 0.85954 0.08909 0.07794 0.72711 0.73446 0.54596 0.54295 151 151 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk unknown unknown   United States 2023-12-01 Undetermined Undetermined Eye Sensory System
29095 29095 SRR27010842 SRX22703625 SRS19696100 SRP475450 PRJNA1047749 Differential expression in Muller glia of injured zebrafish in a mycb / context GSE249116 Transcriptome Analysis This experiment studies retinal regeneration using a zebrafish model with a focus on Muller glia reprogramming. Overall design: Three groups: controls Muller glia from uninjured retinas injured WT Muller glia from injured WT retina at 2 xxx post injury dpi and injured mycb / Muller glia from injured mycb / fish at 2dpi. parent bioproject:PRJNA1047494 pubmed:38984586   Muller glia from WT retinas 2 xxx post injury dpi rep1 [7131 ML 4] [7131 ML 4] GSM7926873   source name:WT Muller glia injured retina|tissue:WT Muller glia injured retina|genotype:WT|treatment:n1|geo loc name:missing|collection date:missing Muller glia from WT retinas 2 xxx post injury dpi rep1 [7131 ML 4] [7131 ML 4] University of Michigan Bioinformatics Core RNA seq pipeline "Watermelon" version 2.4.4 Sequence reads were trimmed for adaptor sequence/low quality sequence using Cutadapt v2.3. FastQC v0.11.8 was used to ensure the quality of data Trimmed sequence reads were mapped to GRCh38/hs19 using STAR v2.7.8a Quantitative count estimates to genes performed with RSEM v1.3.3 Assembly: GRCz11 Supplementary files format and content: RS11 deseq2 raw counts.txt Supplementary files format and content: tab delimited text with include expected counts values for each Sample WT Muller glia injured retina Needle poke injury to retina. Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   tissue:WT Muller glia injured retina|genotype:WT|treatment:n1 GSM7926873 GSM7926873: Muller glia from WT retinas 2 xxx post injury dpi rep1 [7131 ML 4] [7131 ML 4]; Danio rerio; RNA Seq GSM7926873 r1 GSM7926873 1 Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP475450   loader:fastq load.py 7131-ML-4_TTAACGGC-CTCCGTCT_S70_R1_001.fastq.gz 7131-ML-4_TTAACGGC-CTCCGTCT_S70_R2_001.fastq.gz fastq fastq 36567606580.0 121084790.0 GSM7926873 r1 0:151 1:151 A:9477563071;C:8136361552;G:9699876329;T:9253435137;N:370491 151 151     9477563071 8136361552 9699876329 9253435137 370491 SRX22703625 SRS19696100 SRA1761650 University of Michigan University of Michigan 2 0.85059 0.82538 0.08689 0.07693 0.73574 0.74371 0.54735 0.54664 151 151 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk unknown unknown   United States 2023-12-01 Undetermined Undetermined Eye Sensory System
29096 29096 SRR27010843 SRX22703624 SRS19696099 SRP475450 PRJNA1047749 Differential expression in Muller glia of injured zebrafish in a mycb / context GSE249116 Transcriptome Analysis This experiment studies retinal regeneration using a zebrafish model with a focus on Muller glia reprogramming. Overall design: Three groups: controls Muller glia from uninjured retinas injured WT Muller glia from injured WT retina at 2 xxx post injury dpi and injured mycb / Muller glia from injured mycb / fish at 2dpi. parent bioproject:PRJNA1047494 pubmed:38984586   Muller glia from uninjured WT retinas rep2 [7131 ML 2] [7131 ML 2] GSM7926872   source name:WT Muller retinal glia|tissue:WT Muller retinal glia|genotype:WT|treatment:n1|geo loc name:missing|collection date:missing Muller glia from uninjured WT retinas rep2 [7131 ML 2] [7131 ML 2] University of Michigan Bioinformatics Core RNA seq pipeline "Watermelon" version 2.4.4 Sequence reads were trimmed for adaptor sequence/low quality sequence using Cutadapt v2.3. FastQC v0.11.8 was used to ensure the quality of data Trimmed sequence reads were mapped to GRCh38/hs19 using STAR v2.7.8a Quantitative count estimates to genes performed with RSEM v1.3.3 Assembly: GRCz11 Supplementary files format and content: RS11 deseq2 raw counts.txt Supplementary files format and content: tab delimited text with include expected counts values for each Sample WT Muller retinal glia Needle poke injury to retina. Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   tissue:WT Muller retinal glia|genotype:WT|treatment:n1 GSM7926872 GSM7926872: Muller glia from uninjured WT retinas rep2 [7131 ML 2] [7131 ML 2]; Danio rerio; RNA Seq GSM7926872 r1 GSM7926872 1 Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP475450   loader:fastq load.py 7131-ML-2_ACAGAGAA-GCGGTTAC_S68_R1_001.fastq.gz 7131-ML-2_ACAGAGAA-GCGGTTAC_S68_R2_001.fastq.gz fastq fastq 31134149118.0 103093209.0 GSM7926872 r1 0:151 1:151 A:8551703439;C:6621403525;G:7636841163;T:8323884132;N:316859 151 151     8551703439 6621403525 7636841163 8323884132 316859 SRX22703624 SRS19696099 SRA1761650 University of Michigan University of Michigan 2 0.89461 0.85335 0.11633 0.10042 0.74302 0.74943 0.5613 0.54558 151 151 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk unknown unknown   United States 2023-12-01 Undetermined Undetermined Eye Sensory System
29097 29097 SRR27010844 SRX22703623 SRS19696098 SRP475450 PRJNA1047749 Differential expression in Muller glia of injured zebrafish in a mycb / context GSE249116 Transcriptome Analysis This experiment studies retinal regeneration using a zebrafish model with a focus on Muller glia reprogramming. Overall design: Three groups: controls Muller glia from uninjured retinas injured WT Muller glia from injured WT retina at 2 xxx post injury dpi and injured mycb / Muller glia from injured mycb / fish at 2dpi. parent bioproject:PRJNA1047494 pubmed:38984586   Muller glia from uninjured WT retinas rep1 [7131 ML 1] [7131 ML 1] GSM7926871   source name:WT Muller retinal glia|tissue:WT Muller retinal glia|genotype:WT|treatment:n1|geo loc name:missing|collection date:missing Muller glia from uninjured WT retinas rep1 [7131 ML 1] [7131 ML 1] University of Michigan Bioinformatics Core RNA seq pipeline "Watermelon" version 2.4.4 Sequence reads were trimmed for adaptor sequence/low quality sequence using Cutadapt v2.3. FastQC v0.11.8 was used to ensure the quality of data Trimmed sequence reads were mapped to GRCh38/hs19 using STAR v2.7.8a Quantitative count estimates to genes performed with RSEM v1.3.3 Assembly: GRCz11 Supplementary files format and content: RS11 deseq2 raw counts.txt Supplementary files format and content: tab delimited text with include expected counts values for each Sample WT Muller retinal glia Needle poke injury to retina. Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   tissue:WT Muller retinal glia|genotype:WT|treatment:n1 GSM7926871 GSM7926871: Muller glia from uninjured WT retinas rep1 [7131 ML 1] [7131 ML 1]; Danio rerio; RNA Seq GSM7926871 r1 GSM7926871 1 Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP475450   loader:fastq load.py 7131-ML-1_AATGTTGT-TGCTTACA_S67_R1_001.fastq.gz 7131-ML-1_AATGTTGT-TGCTTACA_S67_R2_001.fastq.gz fastq fastq 30223430972.0 100077586.0 GSM7926871 r1 0:151 1:151 A:8370937172;C:6520369124;G:7146916733;T:8184902603;N:305340 151 151     8370937172 6520369124 7146916733 8184902603 305340 SRX22703623 SRS19696098 SRA1761650 University of Michigan University of Michigan 2 0.91221 0.86081 0.12114 0.10476 0.73164 0.73882 0.54727 0.52951 151 151 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk unknown unknown   United States 2023-12-01 Undetermined Undetermined Eye Sensory System
29098 29098 SRR27010846 SRX22703638 SRS19696113 SRP475451 PRJNA1047750 Differential expression in Muller glia of injured zebrafish in a mych / context GSE249115 Transcriptome Analysis This experiment studies retinal regeneration using a zebrafish model with a focus on Muller glia reprogramming. Overall design: Three groups: controls Muller glia from uninjured retinas injured WT Muller glia from injured WT retina at 2 xxx post injury dpi and injured mych / Muller glia from injured mych / fish at 2dpi. Of interest are the WT injury responsive genes and which injury responsive genes in the WT are differentially expressed in the mych / fish. parent bioproject:PRJNA1047494 pubmed:38984586   Muller glia from mych / retinas 2 xxx post injury dpi rep3 [5803 ML 10] GSM7926870   source name:mych / Muller glia injured retina|tissue:mych / Muller glia injured retina|genotype:mych / |treatment:injured|geo loc name:missing|collection date:missing Muller glia from mych / retinas 2 xxx post injury dpi rep3 [5803 ML 10] University of Michigan Bioinformatics Core RNA seq pipeline "Watermelon" version 2.4.4 Sequence reads were trimmed for adaptor sequence/low quality sequence using Cutadapt v2.3. FastQC v0.11.8 was used to ensure the quality of data Trimmed sequence reads were mapped to GRCh38/hs19 using STAR v2.7.8a Quantitative count estimates to genes performed with RSEM v1.3.3 Assembly: GRCz11 Supplementary files format and content: RS10 deseq2 raw counts.txt Supplementary files format and content: tab delimited text with include expected counts values for each Sample mych / Muller glia injured retina Needle poke injury to retina. Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   tissue:mych / Muller glia injured retina|genotype:mych / |treatment:injured GSM7926870 GSM7926870: Muller glia from mych / retinas 2 xxx post injury dpi rep3 [5803 ML 10]; Danio rerio; RNA Seq GSM7926870 r1 GSM7926870 1 Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP475451   loader:fastq load.py 5803-ML-10_AACAACGG-TTACACCT_S222_R1_001.fastq.gz 5803-ML-10_AACAACGG-TTACACCT_S222_R2_001.fastq.gz fastq fastq 33066899590.0 109493045.0 GSM7926870 r1 0:151 1:151 A:9045925528;C:7063243555;G:8277253275;T:8680404068;N:73164 151 151     9045925528 7063243555 8277253275 8680404068 73164 SRX22703638 SRS19696113 SRA1761648 University of Michigan University of Michigan 2 0.87174 0.82563 0.12217 0.10466 0.7052 0.71311 0.50335 0.51416 151 151 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk unknown unknown   United States 2023-12-01 Undetermined Undetermined Eye Sensory System
29099 29099 SRR27010847 SRX22703637 SRS19696112 SRP475451 PRJNA1047750 Differential expression in Muller glia of injured zebrafish in a mych / context GSE249115 Transcriptome Analysis This experiment studies retinal regeneration using a zebrafish model with a focus on Muller glia reprogramming. Overall design: Three groups: controls Muller glia from uninjured retinas injured WT Muller glia from injured WT retina at 2 xxx post injury dpi and injured mych / Muller glia from injured mych / fish at 2dpi. Of interest are the WT injury responsive genes and which injury responsive genes in the WT are differentially expressed in the mych / fish. parent bioproject:PRJNA1047494 pubmed:38984586   Muller glia from mych / retinas 2 xxx post injury dpi rep2 [5803 ML 8] GSM7926869   source name:mych / Muller glia injured retina|tissue:mych / Muller glia injured retina|genotype:mych / |treatment:injured|geo loc name:missing|collection date:missing Muller glia from mych / retinas 2 xxx post injury dpi rep2 [5803 ML 8] University of Michigan Bioinformatics Core RNA seq pipeline "Watermelon" version 2.4.4 Sequence reads were trimmed for adaptor sequence/low quality sequence using Cutadapt v2.3. FastQC v0.11.8 was used to ensure the quality of data Trimmed sequence reads were mapped to GRCh38/hs19 using STAR v2.7.8a Quantitative count estimates to genes performed with RSEM v1.3.3 Assembly: GRCz11 Supplementary files format and content: RS10 deseq2 raw counts.txt Supplementary files format and content: tab delimited text with include expected counts values for each Sample mych / Muller glia injured retina Needle poke injury to retina. Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   tissue:mych / Muller glia injured retina|genotype:mych / |treatment:injured GSM7926869 GSM7926869: Muller glia from mych / retinas 2 xxx post injury dpi rep2 [5803 ML 8]; Danio rerio; RNA Seq GSM7926869 r1 GSM7926869 1 Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP475451   loader:fastq load.py 5803-ML-8_CGAGGTGT-CGCCGTTG_S221_R1_001.fastq.gz 5803-ML-8_CGAGGTGT-CGCCGTTG_S221_R2_001.fastq.gz fastq fastq 45801257486.0 151659793.0 GSM7926869 r1 0:151 1:151 A:12163743496;C:9556065193;G:12415783742;T:11665560316;N:104739 151 151     12163743496 9556065193 12415783742 11665560316 104739 SRX22703637 SRS19696112 SRA1761648 University of Michigan University of Michigan 2 0.82361 0.79223 0.08967 0.0781 0.72614 0.73214 0.51241 0.53445 151 151 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk unknown unknown   United States 2023-12-01 Undetermined Undetermined Eye Sensory System
29100 29100 SRR27010848 SRX22703636 SRS19696111 SRP475451 PRJNA1047750 Differential expression in Muller glia of injured zebrafish in a mych / context GSE249115 Transcriptome Analysis This experiment studies retinal regeneration using a zebrafish model with a focus on Muller glia reprogramming. Overall design: Three groups: controls Muller glia from uninjured retinas injured WT Muller glia from injured WT retina at 2 xxx post injury dpi and injured mych / Muller glia from injured mych / fish at 2dpi. Of interest are the WT injury responsive genes and which injury responsive genes in the WT are differentially expressed in the mych / fish. parent bioproject:PRJNA1047494 pubmed:38984586   Muller glia from mych / retinas 2 xxx post injury dpi rep1 [5803 ML 7] GSM7926868   source name:mych / Muller glia injured retina|tissue:mych / Muller glia injured retina|genotype:mych / |treatment:injured|geo loc name:missing|collection date:missing Muller glia from mych / retinas 2 xxx post injury dpi rep1 [5803 ML 7] University of Michigan Bioinformatics Core RNA seq pipeline "Watermelon" version 2.4.4 Sequence reads were trimmed for adaptor sequence/low quality sequence using Cutadapt v2.3. FastQC v0.11.8 was used to ensure the quality of data Trimmed sequence reads were mapped to GRCh38/hs19 using STAR v2.7.8a Quantitative count estimates to genes performed with RSEM v1.3.3 Assembly: GRCz11 Supplementary files format and content: RS10 deseq2 raw counts.txt Supplementary files format and content: tab delimited text with include expected counts values for each Sample mych / Muller glia injured retina Needle poke injury to retina. Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   tissue:mych / Muller glia injured retina|genotype:mych / |treatment:injured GSM7926868 GSM7926868: Muller glia from mych / retinas 2 xxx post injury dpi rep1 [5803 ML 7]; Danio rerio; RNA Seq GSM7926868 r1 GSM7926868 1 Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP475451   loader:fastq load.py 5803-ML-7_ACCTCCGC-AACCTTCA_S220_R1_001.fastq.gz 5803-ML-7_ACCTCCGC-AACCTTCA_S220_R2_001.fastq.gz fastq fastq 34323078556.0 113652578.0 GSM7926868 r1 0:151 1:151 A:9212407551;C:7253133739;G:9084057760;T:8773400927;N:78579 151 151     9212407551 7253133739 9084057760 8773400927 78579 SRX22703636 SRS19696111 SRA1761648 University of Michigan University of Michigan 2 0.83258 0.79995 0.10182 0.08869 0.7204 0.72506 0.53438 0.53893 151 151 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk unknown unknown   United States 2023-12-01 Undetermined Undetermined Eye Sensory System
29101 29101 SRR27010849 SRX22703635 SRS19696110 SRP475451 PRJNA1047750 Differential expression in Muller glia of injured zebrafish in a mych / context GSE249115 Transcriptome Analysis This experiment studies retinal regeneration using a zebrafish model with a focus on Muller glia reprogramming. Overall design: Three groups: controls Muller glia from uninjured retinas injured WT Muller glia from injured WT retina at 2 xxx post injury dpi and injured mych / Muller glia from injured mych / fish at 2dpi. Of interest are the WT injury responsive genes and which injury responsive genes in the WT are differentially expressed in the mych / fish. parent bioproject:PRJNA1047494 pubmed:38984586   Muller glia from WT retinas 2 xxx post injury dpi rep3 [5803 ML 6] GSM7926867   source name:WT Muller glia injured retina|tissue:WT Muller glia injured retina|genotype:WT|treatment:injured|geo loc name:missing|collection date:missing Muller glia from WT retinas 2 xxx post injury dpi rep3 [5803 ML 6] University of Michigan Bioinformatics Core RNA seq pipeline "Watermelon" version 2.4.4 Sequence reads were trimmed for adaptor sequence/low quality sequence using Cutadapt v2.3. FastQC v0.11.8 was used to ensure the quality of data Trimmed sequence reads were mapped to GRCh38/hs19 using STAR v2.7.8a Quantitative count estimates to genes performed with RSEM v1.3.3 Assembly: GRCz11 Supplementary files format and content: RS10 deseq2 raw counts.txt Supplementary files format and content: tab delimited text with include expected counts values for each Sample WT Muller glia injured retina Needle poke injury to retina. Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   tissue:WT Muller glia injured retina|genotype:WT|treatment:injured GSM7926867 GSM7926867: Muller glia from WT retinas 2 xxx post injury dpi rep3 [5803 ML 6]; Danio rerio; RNA Seq GSM7926867 r1 GSM7926867 1 Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP475451   loader:fastq load.py 5803-ML-6_CTTGAAGG-TAGCGGAG_S219_R1_001.fastq.gz 5803-ML-6_CTTGAAGG-TAGCGGAG_S219_R2_001.fastq.gz fastq fastq 39633900160.0 131238080.0 GSM7926867 r1 0:151 1:151 A:10574730340;C:8244722619;G:10678580933;T:10135778012;N:88256 151 151     10574730340 8244722619 10678580933 10135778012 88256 SRX22703635 SRS19696110 SRA1761648 University of Michigan University of Michigan 2 0.82908 0.8023 0.08841 0.07628 0.72608 0.73285 0.53006 0.52227 151 151 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk unknown unknown   United States 2023-12-01 Undetermined Undetermined Eye Sensory System
29102 29102 SRR27010850 SRX22703634 SRS19696109 SRP475451 PRJNA1047750 Differential expression in Muller glia of injured zebrafish in a mych / context GSE249115 Transcriptome Analysis This experiment studies retinal regeneration using a zebrafish model with a focus on Muller glia reprogramming. Overall design: Three groups: controls Muller glia from uninjured retinas injured WT Muller glia from injured WT retina at 2 xxx post injury dpi and injured mych / Muller glia from injured mych / fish at 2dpi. Of interest are the WT injury responsive genes and which injury responsive genes in the WT are differentially expressed in the mych / fish. parent bioproject:PRJNA1047494 pubmed:38984586   Muller glia from WT retinas 2 xxx post injury dpi rep2 [5803 ML 5] GSM7926866   source name:WT Muller glia injured retina|tissue:WT Muller glia injured retina|genotype:WT|treatment:injured|geo loc name:missing|collection date:missing Muller glia from WT retinas 2 xxx post injury dpi rep2 [5803 ML 5] University of Michigan Bioinformatics Core RNA seq pipeline "Watermelon" version 2.4.4 Sequence reads were trimmed for adaptor sequence/low quality sequence using Cutadapt v2.3. FastQC v0.11.8 was used to ensure the quality of data Trimmed sequence reads were mapped to GRCh38/hs19 using STAR v2.7.8a Quantitative count estimates to genes performed with RSEM v1.3.3 Assembly: GRCz11 Supplementary files format and content: RS10 deseq2 raw counts.txt Supplementary files format and content: tab delimited text with include expected counts values for each Sample WT Muller glia injured retina Needle poke injury to retina. Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   tissue:WT Muller glia injured retina|genotype:WT|treatment:injured GSM7926866 GSM7926866: Muller glia from WT retinas 2 xxx post injury dpi rep2 [5803 ML 5]; Danio rerio; RNA Seq GSM7926866 r1 GSM7926866 1 Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP475451   loader:fastq load.py 5803-ML-5_TCTCGGAG-CCGTGTGT_S218_R1_001.fastq.gz 5803-ML-5_TCTCGGAG-CCGTGTGT_S218_R2_001.fastq.gz fastq fastq 42813650610.0 141767055.0 GSM7926866 r1 0:151 1:151 A:11307756303;C:9128581274;G:11491585338;T:10885631489;N:96206 151 151     11307756303 9128581274 11491585338 10885631489 96206 SRX22703634 SRS19696109 SRA1761648 University of Michigan University of Michigan 2 0.84717 0.81641 0.07575 0.06577 0.72456 0.72947 0.51796 0.52376 151 151 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk unknown unknown   United States 2023-12-01 Undetermined Undetermined Eye Sensory System
29103 29103 SRR27010851 SRX22703633 SRS19696108 SRP475451 PRJNA1047750 Differential expression in Muller glia of injured zebrafish in a mych / context GSE249115 Transcriptome Analysis This experiment studies retinal regeneration using a zebrafish model with a focus on Muller glia reprogramming. Overall design: Three groups: controls Muller glia from uninjured retinas injured WT Muller glia from injured WT retina at 2 xxx post injury dpi and injured mych / Muller glia from injured mych / fish at 2dpi. Of interest are the WT injury responsive genes and which injury responsive genes in the WT are differentially expressed in the mych / fish. parent bioproject:PRJNA1047494 pubmed:38984586   Muller glia from WT retinas 2 xxx post injury dpi rep1 [5803 ML 4] GSM7926865   source name:WT Muller glia injured retina|tissue:WT Muller glia injured retina|genotype:WT|treatment:injured|geo loc name:missing|collection date:missing Muller glia from WT retinas 2 xxx post injury dpi rep1 [5803 ML 4] University of Michigan Bioinformatics Core RNA seq pipeline "Watermelon" version 2.4.4 Sequence reads were trimmed for adaptor sequence/low quality sequence using Cutadapt v2.3. FastQC v0.11.8 was used to ensure the quality of data Trimmed sequence reads were mapped to GRCh38/hs19 using STAR v2.7.8a Quantitative count estimates to genes performed with RSEM v1.3.3 Assembly: GRCz11 Supplementary files format and content: RS10 deseq2 raw counts.txt Supplementary files format and content: tab delimited text with include expected counts values for each Sample WT Muller glia injured retina Needle poke injury to retina. Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   tissue:WT Muller glia injured retina|genotype:WT|treatment:injured GSM7926865 GSM7926865: Muller glia from WT retinas 2 xxx post injury dpi rep1 [5803 ML 4]; Danio rerio; RNA Seq GSM7926865 r1 GSM7926865 1 Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP475451   loader:fastq load.py 5803-ML-4_TTACACAC-AACTCCGA_S217_R1_001.fastq.gz 5803-ML-4_TTACACAC-AACTCCGA_S217_R2_001.fastq.gz fastq fastq 35641811084.0 118019242.0 GSM7926865 r1 0:151 1:151 A:9509123801;C:7547711759;G:9554254572;T:9030640032;N:80920 151 151     9509123801 7547711759 9554254572 9030640032 80920 SRX22703633 SRS19696108 SRA1761648 University of Michigan University of Michigan 2 0.84325 0.81491 0.09102 0.08021 0.71936 0.72368 0.52206 0.51772 151 151 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk unknown unknown   United States 2023-12-01 Undetermined Undetermined Eye Sensory System
29104 29104 SRR27010852 SRX22703632 SRS19696107 SRP475451 PRJNA1047750 Differential expression in Muller glia of injured zebrafish in a mych / context GSE249115 Transcriptome Analysis This experiment studies retinal regeneration using a zebrafish model with a focus on Muller glia reprogramming. Overall design: Three groups: controls Muller glia from uninjured retinas injured WT Muller glia from injured WT retina at 2 xxx post injury dpi and injured mych / Muller glia from injured mych / fish at 2dpi. Of interest are the WT injury responsive genes and which injury responsive genes in the WT are differentially expressed in the mych / fish. parent bioproject:PRJNA1047494 pubmed:38984586   Muller glia from uninjured WT retinas rep3 [5803 ML 3] GSM7926864   source name:WT Muller glia uninjured retina|tissue:WT Muller glia uninjured retina|genotype:WT|treatment:n1|geo loc name:missing|collection date:missing Muller glia from uninjured WT retinas rep3 [5803 ML 3] University of Michigan Bioinformatics Core RNA seq pipeline "Watermelon" version 2.4.4 Sequence reads were trimmed for adaptor sequence/low quality sequence using Cutadapt v2.3. FastQC v0.11.8 was used to ensure the quality of data Trimmed sequence reads were mapped to GRCh38/hs19 using STAR v2.7.8a Quantitative count estimates to genes performed with RSEM v1.3.3 Assembly: GRCz11 Supplementary files format and content: RS10 deseq2 raw counts.txt Supplementary files format and content: tab delimited text with include expected counts values for each Sample WT Muller glia uninjured retina Needle poke injury to retina. Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   tissue:WT Muller glia uninjured retina|genotype:WT|treatment:n1 GSM7926864 GSM7926864: Muller glia from uninjured WT retinas rep3 [5803 ML 3]; Danio rerio; RNA Seq GSM7926864 r1 GSM7926864 1 Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP475451   loader:fastq load.py 5803-ML-3_CCTTATGG-GCACGCTT_S216_R2_001.fastq.gz 5803-ML-3_CCTTATGG-GCACGCTT_S216_R1_001.fastq.gz fastq fastq 44024352302.0 145776001.0 GSM7926864 r1 0:151 1:151 A:12035679681;C:9067037632;G:11325935498;T:11595599635;N:99856 151 151     12035679681 9067037632 11325935498 11595599635 99856 SRX22703632 SRS19696107 SRA1761648 University of Michigan University of Michigan 2 0.83165 0.79093 0.139 0.11939 0.73539 0.74148 0.52839 0.52569 151 151 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk unknown unknown   United States 2023-12-01 Undetermined Undetermined Eye Sensory System
29105 29105 SRR27010853 SRX22703631 SRS19696106 SRP475451 PRJNA1047750 Differential expression in Muller glia of injured zebrafish in a mych / context GSE249115 Transcriptome Analysis This experiment studies retinal regeneration using a zebrafish model with a focus on Muller glia reprogramming. Overall design: Three groups: controls Muller glia from uninjured retinas injured WT Muller glia from injured WT retina at 2 xxx post injury dpi and injured mych / Muller glia from injured mych / fish at 2dpi. Of interest are the WT injury responsive genes and which injury responsive genes in the WT are differentially expressed in the mych / fish. parent bioproject:PRJNA1047494 pubmed:38984586   Muller glia from uninjured WT retinas rep2 [5803 ML 2] GSM7926863   source name:WT Muller glia uninjured retina|tissue:WT Muller glia uninjured retina|genotype:WT|treatment:n1|geo loc name:missing|collection date:missing Muller glia from uninjured WT retinas rep2 [5803 ML 2] University of Michigan Bioinformatics Core RNA seq pipeline "Watermelon" version 2.4.4 Sequence reads were trimmed for adaptor sequence/low quality sequence using Cutadapt v2.3. FastQC v0.11.8 was used to ensure the quality of data Trimmed sequence reads were mapped to GRCh38/hs19 using STAR v2.7.8a Quantitative count estimates to genes performed with RSEM v1.3.3 Assembly: GRCz11 Supplementary files format and content: RS10 deseq2 raw counts.txt Supplementary files format and content: tab delimited text with include expected counts values for each Sample WT Muller glia uninjured retina Needle poke injury to retina. Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   tissue:WT Muller glia uninjured retina|genotype:WT|treatment:n1 GSM7926863 GSM7926863: Muller glia from uninjured WT retinas rep2 [5803 ML 2]; Danio rerio; RNA Seq GSM7926863 r1 GSM7926863 1 Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP475451   loader:fastq load.py 5803-ML-2_AGGTCTAT-CCGTGAGA_S215_R1_001.fastq.gz 5803-ML-2_AGGTCTAT-CCGTGAGA_S215_R2_001.fastq.gz fastq fastq 30776465150.0 101908825.0 GSM7926863 r1 0:151 1:151 A:8433807492;C:6356747786;G:7933043571;T:8052796793;N:69508 151 151     8433807492 6356747786 7933043571 8052796793 69508 SRX22703631 SRS19696106 SRA1761648 University of Michigan University of Michigan 2 0.84084 0.79586 0.14119 0.12143 0.74067 0.74718 0.52859 0.5313 151 151 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk unknown unknown   United States 2023-12-01 Undetermined Undetermined Eye Sensory System
29106 29106 SRR27010854 SRX22703630 SRS19696105 SRP475451 PRJNA1047750 Differential expression in Muller glia of injured zebrafish in a mych / context GSE249115 Transcriptome Analysis This experiment studies retinal regeneration using a zebrafish model with a focus on Muller glia reprogramming. Overall design: Three groups: controls Muller glia from uninjured retinas injured WT Muller glia from injured WT retina at 2 xxx post injury dpi and injured mych / Muller glia from injured mych / fish at 2dpi. Of interest are the WT injury responsive genes and which injury responsive genes in the WT are differentially expressed in the mych / fish. parent bioproject:PRJNA1047494 pubmed:38984586   Muller glia from uninjured WT retinas rep1 [5803 ML 1 ] GSM7926862   source name:WT Muller glia uninjured retina|tissue:WT Muller glia uninjured retina|genotype:WT|treatment:n1|geo loc name:missing|collection date:missing Muller glia from uninjured WT retinas rep1 [5803 ML 1 ] University of Michigan Bioinformatics Core RNA seq pipeline "Watermelon" version 2.4.4 Sequence reads were trimmed for adaptor sequence/low quality sequence using Cutadapt v2.3. FastQC v0.11.8 was used to ensure the quality of data Trimmed sequence reads were mapped to GRCh38/hs19 using STAR v2.7.8a Quantitative count estimates to genes performed with RSEM v1.3.3 Assembly: GRCz11 Supplementary files format and content: RS10 deseq2 raw counts.txt Supplementary files format and content: tab delimited text with include expected counts values for each Sample WT Muller glia uninjured retina Needle poke injury to retina. Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   tissue:WT Muller glia uninjured retina|genotype:WT|treatment:n1 GSM7926862 GSM7926862: Muller glia from uninjured WT retinas rep1 [5803 ML 1 ]; Danio rerio; RNA Seq GSM7926862 r1 GSM7926862 1 Retinas from gfap:GFP transgenic zebrafish were dissected from fish eyes tissue was dissociated using Papain Dissociation system Worthington Biochemical and GFP+ Muller glia isolated by FACS. RNA was purified using Directzol RNA microprep kit Zymo Research. Illumina RNA PolyA enrichment library prep kit was used according to manufacturer's directions.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP475451   loader:fastq load.py 5803-ML-1_AAGGACCG-GACACAAG_S214_R1_001.fastq.gz 5803-ML-1_AAGGACCG-GACACAAG_S214_R2_001.fastq.gz fastq fastq 35814764672.0 118591936.0 GSM7926862 r1 0:151 1:151 A:10118857494;C:7312994344;G:8767136259;T:9615695615;N:80960 151 151     10118857494 7312994344 8767136259 9615695615 80960 SRX22703630 SRS19696105 SRA1761648 University of Michigan University of Michigan 2 0.84915 0.79824 0.17335 0.14475 0.72103 0.73097 0.51291 0.4893 151 151 B B biological fallback assumption illumina novaseq_era unknown poly_a unknown bulk unknown unknown   United States 2023-12-01 Undetermined Undetermined Eye Sensory System
30572 30572 SRR27848813 SRX23511754 SRS20362222 SRP487850 PRJNA1072679 Oxidative stress altered glucose metabolism and inflammation contribute to the retinal phenotype in the choroideremia zebrafish GSE254948 Transcriptome Analysis Reactive oxygen species ROS within the retina play a key role in maintaining function and cell survival. However excessive ROS can lead to oxidative stress inducing dysregulation of metabolic and inflammatory pathways. The chmru848 zebrafish models choroideremia CHM an X linked chorioretinal dystrophy which predominantly affects the photoreceptors retinal pigment epithelium RPE and choroid. In this study we examined the transcriptomic signature of the chmru848 zebrafish retina to reveal upregulation of cytokine pathways and glia migration upregulation of oxidative ER stress and apoptosis markers and dysregulation of glucose metabolism with downregulation of glycolysis and upregulation of the oxidative phase of the pentose phosphate pathway. Glucose uptake was overall impaired in the chmru848 retina using the 2 NBDG glucose uptake assay but post overexpression of human PFKM it partially rescued the retinal phenotype and glucose uptake but without xxx the expression of glycolysis markers. Therapies targeting glucose metabolism in CHM may represent a potential remedial approach. Overall design: Choroideremia CHM is a x linked inherited retinal dystrophy caused by mutations in the CHM gene. It is a progressive condition leading to complete blindness. We are working on a chm zebrafish model which shows a widespread severe degenerative phenotype with embryos only surviving up to 5 days. We compared the transcriptome in wildtype vs chm zebrafish retinas and determine which pathways are disrupted in chm fish.       Wildtype 7 GSM8061015   source name:Retina|tissue:Retina|genotype:Wildtype|geo loc name:missing|collection date:missing Wildtype 7 Raw reads were quality and adapter trimmed using trimgalore version 0.6.7 14 before alignment. Reads were mapped and subsequent gene level counted using RSEM 1.3.3 15 and STAR 2.7.10a 16 against the zebrafish genome GRCz11 both from Ensembl. Normalisation of raw count data and differential expression analysis was performed with the DESeq2 package version 1.38.3within the R programming environment version 4.2.2. Assembly: GRCz11 Supplementary files format and content: VST normalised expression values. Rows = genes; Columns = samples. Retina   Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   tissue:Retina|genotype:Wildtype GSM8061015 GSM8061015: Wildtype 7; Danio rerio; RNA Seq GSM8061015 r1 GSM8061015 1 Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP487850   loader:fastq load.py WT7_1.fq.gz WT7_2.fq.gz fastq fastq 16387213200.0 54624044.0 GSM8061015 r1 0:150 1:150 A:4787207224;C:3312386758;G:3384738324;T:4902574775;N:306119 150 150     4787207224 3312386758 3384738324 4902574775 306119 SRX23511754 SRS20362222 SRA1796957 The Francis Crick Institute The Francis Crick Institute 2 0.92431 0.91825 0.16903 0.16976 0.74067 0.75189 0.50167 0.50261 150 150 B B biological fallback assumption illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_plate smartseq   United Kingdom 2024-02-02 Undetermined Embryo Eye Sensory System
30573 30573 SRR27848814 SRX23511753 SRS20362221 SRP487850 PRJNA1072679 Oxidative stress altered glucose metabolism and inflammation contribute to the retinal phenotype in the choroideremia zebrafish GSE254948 Transcriptome Analysis Reactive oxygen species ROS within the retina play a key role in maintaining function and cell survival. However excessive ROS can lead to oxidative stress inducing dysregulation of metabolic and inflammatory pathways. The chmru848 zebrafish models choroideremia CHM an X linked chorioretinal dystrophy which predominantly affects the photoreceptors retinal pigment epithelium RPE and choroid. In this study we examined the transcriptomic signature of the chmru848 zebrafish retina to reveal upregulation of cytokine pathways and glia migration upregulation of oxidative ER stress and apoptosis markers and dysregulation of glucose metabolism with downregulation of glycolysis and upregulation of the oxidative phase of the pentose phosphate pathway. Glucose uptake was overall impaired in the chmru848 retina using the 2 NBDG glucose uptake assay but post overexpression of human PFKM it partially rescued the retinal phenotype and glucose uptake but without xxx the expression of glycolysis markers. Therapies targeting glucose metabolism in CHM may represent a potential remedial approach. Overall design: Choroideremia CHM is a x linked inherited retinal dystrophy caused by mutations in the CHM gene. It is a progressive condition leading to complete blindness. We are working on a chm zebrafish model which shows a widespread severe degenerative phenotype with embryos only surviving up to 5 days. We compared the transcriptome in wildtype vs chm zebrafish retinas and determine which pathways are disrupted in chm fish.       Wildtype 6 GSM8061014   source name:Retina|tissue:Retina|genotype:Wildtype|geo loc name:missing|collection date:missing Wildtype 6 Raw reads were quality and adapter trimmed using trimgalore version 0.6.7 14 before alignment. Reads were mapped and subsequent gene level counted using RSEM 1.3.3 15 and STAR 2.7.10a 16 against the zebrafish genome GRCz11 both from Ensembl. Normalisation of raw count data and differential expression analysis was performed with the DESeq2 package version 1.38.3within the R programming environment version 4.2.2. Assembly: GRCz11 Supplementary files format and content: VST normalised expression values. Rows = genes; Columns = samples. Retina   Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   tissue:Retina|genotype:Wildtype GSM8061014 GSM8061014: Wildtype 6; Danio rerio; RNA Seq GSM8061014 r1 GSM8061014 1 Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP487850   loader:fastq load.py WT6_1.fq.gz WT6_2.fq.gz fastq fastq 19114278900.0 63714263.0 GSM8061014 r1 0:150 1:150 A:5885874877;C:3513555230;G:3610119151;T:6104379853;N:349789 150 150     5885874877 3513555230 3610119151 6104379853 349789 SRX23511753 SRS20362221 SRA1796957 The Francis Crick Institute The Francis Crick Institute 2 0.88104 0.87237 0.33066 0.33508 0.7357 0.7499 0.52706 0.51369 150 150 B B biological fallback assumption illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_plate smartseq   United Kingdom 2024-02-02 Undetermined Embryo Eye Sensory System
30574 30574 SRR27848815 SRX23511752 SRS20362220 SRP487850 PRJNA1072679 Oxidative stress altered glucose metabolism and inflammation contribute to the retinal phenotype in the choroideremia zebrafish GSE254948 Transcriptome Analysis Reactive oxygen species ROS within the retina play a key role in maintaining function and cell survival. However excessive ROS can lead to oxidative stress inducing dysregulation of metabolic and inflammatory pathways. The chmru848 zebrafish models choroideremia CHM an X linked chorioretinal dystrophy which predominantly affects the photoreceptors retinal pigment epithelium RPE and choroid. In this study we examined the transcriptomic signature of the chmru848 zebrafish retina to reveal upregulation of cytokine pathways and glia migration upregulation of oxidative ER stress and apoptosis markers and dysregulation of glucose metabolism with downregulation of glycolysis and upregulation of the oxidative phase of the pentose phosphate pathway. Glucose uptake was overall impaired in the chmru848 retina using the 2 NBDG glucose uptake assay but post overexpression of human PFKM it partially rescued the retinal phenotype and glucose uptake but without xxx the expression of glycolysis markers. Therapies targeting glucose metabolism in CHM may represent a potential remedial approach. Overall design: Choroideremia CHM is a x linked inherited retinal dystrophy caused by mutations in the CHM gene. It is a progressive condition leading to complete blindness. We are working on a chm zebrafish model which shows a widespread severe degenerative phenotype with embryos only surviving up to 5 days. We compared the transcriptome in wildtype vs chm zebrafish retinas and determine which pathways are disrupted in chm fish.       Wildtype 5 GSM8061013   source name:Retina|tissue:Retina|genotype:Wildtype|geo loc name:missing|collection date:missing Wildtype 5 Raw reads were quality and adapter trimmed using trimgalore version 0.6.7 14 before alignment. Reads were mapped and subsequent gene level counted using RSEM 1.3.3 15 and STAR 2.7.10a 16 against the zebrafish genome GRCz11 both from Ensembl. Normalisation of raw count data and differential expression analysis was performed with the DESeq2 package version 1.38.3within the R programming environment version 4.2.2. Assembly: GRCz11 Supplementary files format and content: VST normalised expression values. Rows = genes; Columns = samples. Retina   Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   tissue:Retina|genotype:Wildtype GSM8061013 GSM8061013: Wildtype 5; Danio rerio; RNA Seq GSM8061013 r1 GSM8061013 1 Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP487850   loader:fastq load.py WT5_2.fq.gz WT5_1.fq.gz fastq fastq 16120305900.0 53734353.0 GSM8061013 r1 0:150 1:150 A:4774586081;C:3152393582;G:3245217813;T:4947808687;N:299737 150 150     4774586081 3152393582 3245217813 4947808687 299737 SRX23511752 SRS20362220 SRA1796957 The Francis Crick Institute The Francis Crick Institute 2 0.90351 0.89372 0.19598 0.19188 0.74468 0.76037 0.49643 0.5012 150 150 B B biological fallback assumption illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_plate smartseq   United Kingdom 2024-02-02 Undetermined Embryo Eye Sensory System
30575 30575 SRR27848816 SRX23511751 SRS20362219 SRP487850 PRJNA1072679 Oxidative stress altered glucose metabolism and inflammation contribute to the retinal phenotype in the choroideremia zebrafish GSE254948 Transcriptome Analysis Reactive oxygen species ROS within the retina play a key role in maintaining function and cell survival. However excessive ROS can lead to oxidative stress inducing dysregulation of metabolic and inflammatory pathways. The chmru848 zebrafish models choroideremia CHM an X linked chorioretinal dystrophy which predominantly affects the photoreceptors retinal pigment epithelium RPE and choroid. In this study we examined the transcriptomic signature of the chmru848 zebrafish retina to reveal upregulation of cytokine pathways and glia migration upregulation of oxidative ER stress and apoptosis markers and dysregulation of glucose metabolism with downregulation of glycolysis and upregulation of the oxidative phase of the pentose phosphate pathway. Glucose uptake was overall impaired in the chmru848 retina using the 2 NBDG glucose uptake assay but post overexpression of human PFKM it partially rescued the retinal phenotype and glucose uptake but without xxx the expression of glycolysis markers. Therapies targeting glucose metabolism in CHM may represent a potential remedial approach. Overall design: Choroideremia CHM is a x linked inherited retinal dystrophy caused by mutations in the CHM gene. It is a progressive condition leading to complete blindness. We are working on a chm zebrafish model which shows a widespread severe degenerative phenotype with embryos only surviving up to 5 days. We compared the transcriptome in wildtype vs chm zebrafish retinas and determine which pathways are disrupted in chm fish.       Wildtype 4 GSM8061012   source name:Retina|tissue:Retina|genotype:Wildtype|geo loc name:missing|collection date:missing Wildtype 4 Raw reads were quality and adapter trimmed using trimgalore version 0.6.7 14 before alignment. Reads were mapped and subsequent gene level counted using RSEM 1.3.3 15 and STAR 2.7.10a 16 against the zebrafish genome GRCz11 both from Ensembl. Normalisation of raw count data and differential expression analysis was performed with the DESeq2 package version 1.38.3within the R programming environment version 4.2.2. Assembly: GRCz11 Supplementary files format and content: VST normalised expression values. Rows = genes; Columns = samples. Retina   Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   tissue:Retina|genotype:Wildtype GSM8061012 GSM8061012: Wildtype 4; Danio rerio; RNA Seq GSM8061012 r1 GSM8061012 1 Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP487850   loader:fastq load.py WT4_1.fq.gz WT4_2.fq.gz fastq fastq 15545813700.0 51819379.0 GSM8061012 r1 0:150 1:150 A:4735339999;C:2883223993;G:2979819364;T:4947148649;N:281695 150 150     4735339999 2883223993 2979819364 4947148649 281695 SRX23511751 SRS20362219 SRA1796957 The Francis Crick Institute The Francis Crick Institute 2 0.88431 0.87409 0.2472 0.24596 0.74675 0.76534 0.50472 0.51348 150 150 B B biological fallback assumption illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_plate smartseq   United Kingdom 2024-02-02 Undetermined Embryo Eye Sensory System
30576 30576 SRR27848817 SRX23511750 SRS20362218 SRP487850 PRJNA1072679 Oxidative stress altered glucose metabolism and inflammation contribute to the retinal phenotype in the choroideremia zebrafish GSE254948 Transcriptome Analysis Reactive oxygen species ROS within the retina play a key role in maintaining function and cell survival. However excessive ROS can lead to oxidative stress inducing dysregulation of metabolic and inflammatory pathways. The chmru848 zebrafish models choroideremia CHM an X linked chorioretinal dystrophy which predominantly affects the photoreceptors retinal pigment epithelium RPE and choroid. In this study we examined the transcriptomic signature of the chmru848 zebrafish retina to reveal upregulation of cytokine pathways and glia migration upregulation of oxidative ER stress and apoptosis markers and dysregulation of glucose metabolism with downregulation of glycolysis and upregulation of the oxidative phase of the pentose phosphate pathway. Glucose uptake was overall impaired in the chmru848 retina using the 2 NBDG glucose uptake assay but post overexpression of human PFKM it partially rescued the retinal phenotype and glucose uptake but without xxx the expression of glycolysis markers. Therapies targeting glucose metabolism in CHM may represent a potential remedial approach. Overall design: Choroideremia CHM is a x linked inherited retinal dystrophy caused by mutations in the CHM gene. It is a progressive condition leading to complete blindness. We are working on a chm zebrafish model which shows a widespread severe degenerative phenotype with embryos only surviving up to 5 days. We compared the transcriptome in wildtype vs chm zebrafish retinas and determine which pathways are disrupted in chm fish.       Wildtype 3 GSM8061011   source name:Retina|tissue:Retina|genotype:Wildtype|geo loc name:missing|collection date:missing Wildtype 3 Raw reads were quality and adapter trimmed using trimgalore version 0.6.7 14 before alignment. Reads were mapped and subsequent gene level counted using RSEM 1.3.3 15 and STAR 2.7.10a 16 against the zebrafish genome GRCz11 both from Ensembl. Normalisation of raw count data and differential expression analysis was performed with the DESeq2 package version 1.38.3within the R programming environment version 4.2.2. Assembly: GRCz11 Supplementary files format and content: VST normalised expression values. Rows = genes; Columns = samples. Retina   Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   tissue:Retina|genotype:Wildtype GSM8061011 GSM8061011: Wildtype 3; Danio rerio; RNA Seq GSM8061011 r1 GSM8061011 1 Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP487850   loader:fastq load.py WT3_1.fq.gz WT3_2.fq.gz fastq fastq 17883593700.0 59611979.0 GSM8061011 r1 0:150 1:150 A:5305029339;C:3503937995;G:3594774195;T:5479523425;N:328746 150 150     5305029339 3503937995 3594774195 5479523425 328746 SRX23511750 SRS20362218 SRA1796957 The Francis Crick Institute The Francis Crick Institute 2 0.90376 0.89786 0.22127 0.22019 0.72941 0.74257 0.49283 0.4977 150 150 B B biological fallback assumption illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_plate smartseq   United Kingdom 2024-02-02 Undetermined Embryo Eye Sensory System
30577 30577 SRR27848818 SRX23511749 SRS20362217 SRP487850 PRJNA1072679 Oxidative stress altered glucose metabolism and inflammation contribute to the retinal phenotype in the choroideremia zebrafish GSE254948 Transcriptome Analysis Reactive oxygen species ROS within the retina play a key role in maintaining function and cell survival. However excessive ROS can lead to oxidative stress inducing dysregulation of metabolic and inflammatory pathways. The chmru848 zebrafish models choroideremia CHM an X linked chorioretinal dystrophy which predominantly affects the photoreceptors retinal pigment epithelium RPE and choroid. In this study we examined the transcriptomic signature of the chmru848 zebrafish retina to reveal upregulation of cytokine pathways and glia migration upregulation of oxidative ER stress and apoptosis markers and dysregulation of glucose metabolism with downregulation of glycolysis and upregulation of the oxidative phase of the pentose phosphate pathway. Glucose uptake was overall impaired in the chmru848 retina using the 2 NBDG glucose uptake assay but post overexpression of human PFKM it partially rescued the retinal phenotype and glucose uptake but without xxx the expression of glycolysis markers. Therapies targeting glucose metabolism in CHM may represent a potential remedial approach. Overall design: Choroideremia CHM is a x linked inherited retinal dystrophy caused by mutations in the CHM gene. It is a progressive condition leading to complete blindness. We are working on a chm zebrafish model which shows a widespread severe degenerative phenotype with embryos only surviving up to 5 days. We compared the transcriptome in wildtype vs chm zebrafish retinas and determine which pathways are disrupted in chm fish.       Wildtype 2 GSM8061010   source name:Retina|tissue:Retina|genotype:Wildtype|geo loc name:missing|collection date:missing Wildtype 2 Raw reads were quality and adapter trimmed using trimgalore version 0.6.7 14 before alignment. Reads were mapped and subsequent gene level counted using RSEM 1.3.3 15 and STAR 2.7.10a 16 against the zebrafish genome GRCz11 both from Ensembl. Normalisation of raw count data and differential expression analysis was performed with the DESeq2 package version 1.38.3within the R programming environment version 4.2.2. Assembly: GRCz11 Supplementary files format and content: VST normalised expression values. Rows = genes; Columns = samples. Retina   Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   tissue:Retina|genotype:Wildtype GSM8061010 GSM8061010: Wildtype 2; Danio rerio; RNA Seq GSM8061010 r1 GSM8061010 1 Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP487850   loader:fastq load.py WT2_1.fq.gz WT2_2.fq.gz fastq fastq 15921002400.0 53070008.0 GSM8061010 r1 0:150 1:150 A:4801739158;C:2898179014;G:3037454009;T:5183338180;N:292039 150 150     4801739158 2898179014 3037454009 5183338180 292039 SRX23511749 SRS20362217 SRA1796957 The Francis Crick Institute The Francis Crick Institute 2 0.88935 0.87616 0.22376 0.2085 0.75367 0.78123 0.53443 0.51122 150 150 B B biological fallback assumption illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_plate smartseq   United Kingdom 2024-02-02 Undetermined Embryo Eye Sensory System
30578 30578 SRR27848819 SRX23511748 SRS20362216 SRP487850 PRJNA1072679 Oxidative stress altered glucose metabolism and inflammation contribute to the retinal phenotype in the choroideremia zebrafish GSE254948 Transcriptome Analysis Reactive oxygen species ROS within the retina play a key role in maintaining function and cell survival. However excessive ROS can lead to oxidative stress inducing dysregulation of metabolic and inflammatory pathways. The chmru848 zebrafish models choroideremia CHM an X linked chorioretinal dystrophy which predominantly affects the photoreceptors retinal pigment epithelium RPE and choroid. In this study we examined the transcriptomic signature of the chmru848 zebrafish retina to reveal upregulation of cytokine pathways and glia migration upregulation of oxidative ER stress and apoptosis markers and dysregulation of glucose metabolism with downregulation of glycolysis and upregulation of the oxidative phase of the pentose phosphate pathway. Glucose uptake was overall impaired in the chmru848 retina using the 2 NBDG glucose uptake assay but post overexpression of human PFKM it partially rescued the retinal phenotype and glucose uptake but without xxx the expression of glycolysis markers. Therapies targeting glucose metabolism in CHM may represent a potential remedial approach. Overall design: Choroideremia CHM is a x linked inherited retinal dystrophy caused by mutations in the CHM gene. It is a progressive condition leading to complete blindness. We are working on a chm zebrafish model which shows a widespread severe degenerative phenotype with embryos only surviving up to 5 days. We compared the transcriptome in wildtype vs chm zebrafish retinas and determine which pathways are disrupted in chm fish.       Wildtype 1 GSM8061009   source name:Retina|tissue:Retina|genotype:Wildtype|geo loc name:missing|collection date:missing Wildtype 1 Raw reads were quality and adapter trimmed using trimgalore version 0.6.7 14 before alignment. Reads were mapped and subsequent gene level counted using RSEM 1.3.3 15 and STAR 2.7.10a 16 against the zebrafish genome GRCz11 both from Ensembl. Normalisation of raw count data and differential expression analysis was performed with the DESeq2 package version 1.38.3within the R programming environment version 4.2.2. Assembly: GRCz11 Supplementary files format and content: VST normalised expression values. Rows = genes; Columns = samples. Retina   Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   tissue:Retina|genotype:Wildtype GSM8061009 GSM8061009: Wildtype 1; Danio rerio; RNA Seq GSM8061009 r1 GSM8061009 1 Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP487850   loader:fastq load.py WT1_1.fq.gz WT1_2.fq.gz fastq fastq 15664926900.0 52216423.0 GSM8061009 r1 0:150 1:150 A:4654413024;C:3082161368;G:3150203371;T:4777859952;N:289185 150 150     4654413024 3082161368 3150203371 4777859952 289185 SRX23511748 SRS20362216 SRA1796957 The Francis Crick Institute The Francis Crick Institute 2 0.9012 0.89646 0.22096 0.21863 0.73507 0.7475 0.4816 0.4726 150 150 B B biological fallback assumption illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_plate smartseq   United Kingdom 2024-02-02 Undetermined Embryo Eye Sensory System
30579 30579 SRR27848820 SRX23511747 SRS20362215 SRP487850 PRJNA1072679 Oxidative stress altered glucose metabolism and inflammation contribute to the retinal phenotype in the choroideremia zebrafish GSE254948 Transcriptome Analysis Reactive oxygen species ROS within the retina play a key role in maintaining function and cell survival. However excessive ROS can lead to oxidative stress inducing dysregulation of metabolic and inflammatory pathways. The chmru848 zebrafish models choroideremia CHM an X linked chorioretinal dystrophy which predominantly affects the photoreceptors retinal pigment epithelium RPE and choroid. In this study we examined the transcriptomic signature of the chmru848 zebrafish retina to reveal upregulation of cytokine pathways and glia migration upregulation of oxidative ER stress and apoptosis markers and dysregulation of glucose metabolism with downregulation of glycolysis and upregulation of the oxidative phase of the pentose phosphate pathway. Glucose uptake was overall impaired in the chmru848 retina using the 2 NBDG glucose uptake assay but post overexpression of human PFKM it partially rescued the retinal phenotype and glucose uptake but without xxx the expression of glycolysis markers. Therapies targeting glucose metabolism in CHM may represent a potential remedial approach. Overall design: Choroideremia CHM is a x linked inherited retinal dystrophy caused by mutations in the CHM gene. It is a progressive condition leading to complete blindness. We are working on a chm zebrafish model which shows a widespread severe degenerative phenotype with embryos only surviving up to 5 days. We compared the transcriptome in wildtype vs chm zebrafish retinas and determine which pathways are disrupted in chm fish.       Choroideremia 7 GSM8061008   source name:Retina|tissue:Retina|genotype:Choroideremia|geo loc name:missing|collection date:missing Choroideremia 7 Raw reads were quality and adapter trimmed using trimgalore version 0.6.7 14 before alignment. Reads were mapped and subsequent gene level counted using RSEM 1.3.3 15 and STAR 2.7.10a 16 against the zebrafish genome GRCz11 both from Ensembl. Normalisation of raw count data and differential expression analysis was performed with the DESeq2 package version 1.38.3within the R programming environment version 4.2.2. Assembly: GRCz11 Supplementary files format and content: VST normalised expression values. Rows = genes; Columns = samples. Retina   Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   tissue:Retina|genotype:Choroideremia GSM8061008 GSM8061008: Choroideremia 7; Danio rerio; RNA Seq GSM8061008 r1 GSM8061008 1 Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP487850   loader:fastq load.py CHM7_2.fq.gz CHM7_1.fq.gz fastq fastq 16991281200.0 56637604.0 GSM8061008 r1 0:150 1:150 A:4896856485;C:3497447652;G:3598783228;T:4998138764;N:55071 150 150     4896856485 3497447652 3598783228 4998138764 55071 SRX23511747 SRS20362215 SRA1796957 The Francis Crick Institute The Francis Crick Institute 2 0.926 0.92518 0.16779 0.16551 0.72393 0.73513 0.51723 0.51214 150 150 B B biological fallback assumption illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_plate smartseq   United Kingdom 2024-02-02 Undetermined Embryo Eye Sensory System
30580 30580 SRR27848821 SRX23511746 SRS20362214 SRP487850 PRJNA1072679 Oxidative stress altered glucose metabolism and inflammation contribute to the retinal phenotype in the choroideremia zebrafish GSE254948 Transcriptome Analysis Reactive oxygen species ROS within the retina play a key role in maintaining function and cell survival. However excessive ROS can lead to oxidative stress inducing dysregulation of metabolic and inflammatory pathways. The chmru848 zebrafish models choroideremia CHM an X linked chorioretinal dystrophy which predominantly affects the photoreceptors retinal pigment epithelium RPE and choroid. In this study we examined the transcriptomic signature of the chmru848 zebrafish retina to reveal upregulation of cytokine pathways and glia migration upregulation of oxidative ER stress and apoptosis markers and dysregulation of glucose metabolism with downregulation of glycolysis and upregulation of the oxidative phase of the pentose phosphate pathway. Glucose uptake was overall impaired in the chmru848 retina using the 2 NBDG glucose uptake assay but post overexpression of human PFKM it partially rescued the retinal phenotype and glucose uptake but without xxx the expression of glycolysis markers. Therapies targeting glucose metabolism in CHM may represent a potential remedial approach. Overall design: Choroideremia CHM is a x linked inherited retinal dystrophy caused by mutations in the CHM gene. It is a progressive condition leading to complete blindness. We are working on a chm zebrafish model which shows a widespread severe degenerative phenotype with embryos only surviving up to 5 days. We compared the transcriptome in wildtype vs chm zebrafish retinas and determine which pathways are disrupted in chm fish.       Choroideremia 6 GSM8061007   source name:Retina|tissue:Retina|genotype:Choroideremia|geo loc name:missing|collection date:missing Choroideremia 6 Raw reads were quality and adapter trimmed using trimgalore version 0.6.7 14 before alignment. Reads were mapped and subsequent gene level counted using RSEM 1.3.3 15 and STAR 2.7.10a 16 against the zebrafish genome GRCz11 both from Ensembl. Normalisation of raw count data and differential expression analysis was performed with the DESeq2 package version 1.38.3within the R programming environment version 4.2.2. Assembly: GRCz11 Supplementary files format and content: VST normalised expression values. Rows = genes; Columns = samples. Retina   Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   tissue:Retina|genotype:Choroideremia GSM8061007 GSM8061007: Choroideremia 6; Danio rerio; RNA Seq GSM8061007 r1 GSM8061007 1 Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP487850   loader:fastq load.py CHM6_1.fq.gz CHM6_2.fq.gz fastq fastq 18881901900.0 62939673.0 GSM8061007 r1 0:150 1:150 A:5796692721;C:3348140904;G:3547934333;T:6189072751;N:61191 150 150     5796692721 3348140904 3547934333 6189072751 61191 SRX23511746 SRS20362214 SRA1796957 The Francis Crick Institute The Francis Crick Institute 2 0.86869 0.86484 0.26053 0.2569 0.75057 0.77705 0.66121 0.65975 150 150 B B biological fallback assumption illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_plate smartseq   United Kingdom 2024-02-02 Undetermined Embryo Eye Sensory System
30581 30581 SRR27848822 SRX23511745 SRS20362213 SRP487850 PRJNA1072679 Oxidative stress altered glucose metabolism and inflammation contribute to the retinal phenotype in the choroideremia zebrafish GSE254948 Transcriptome Analysis Reactive oxygen species ROS within the retina play a key role in maintaining function and cell survival. However excessive ROS can lead to oxidative stress inducing dysregulation of metabolic and inflammatory pathways. The chmru848 zebrafish models choroideremia CHM an X linked chorioretinal dystrophy which predominantly affects the photoreceptors retinal pigment epithelium RPE and choroid. In this study we examined the transcriptomic signature of the chmru848 zebrafish retina to reveal upregulation of cytokine pathways and glia migration upregulation of oxidative ER stress and apoptosis markers and dysregulation of glucose metabolism with downregulation of glycolysis and upregulation of the oxidative phase of the pentose phosphate pathway. Glucose uptake was overall impaired in the chmru848 retina using the 2 NBDG glucose uptake assay but post overexpression of human PFKM it partially rescued the retinal phenotype and glucose uptake but without xxx the expression of glycolysis markers. Therapies targeting glucose metabolism in CHM may represent a potential remedial approach. Overall design: Choroideremia CHM is a x linked inherited retinal dystrophy caused by mutations in the CHM gene. It is a progressive condition leading to complete blindness. We are working on a chm zebrafish model which shows a widespread severe degenerative phenotype with embryos only surviving up to 5 days. We compared the transcriptome in wildtype vs chm zebrafish retinas and determine which pathways are disrupted in chm fish.       Choroideremia 5 GSM8061006   source name:Retina|tissue:Retina|genotype:Choroideremia|geo loc name:missing|collection date:missing Choroideremia 5 Raw reads were quality and adapter trimmed using trimgalore version 0.6.7 14 before alignment. Reads were mapped and subsequent gene level counted using RSEM 1.3.3 15 and STAR 2.7.10a 16 against the zebrafish genome GRCz11 both from Ensembl. Normalisation of raw count data and differential expression analysis was performed with the DESeq2 package version 1.38.3within the R programming environment version 4.2.2. Assembly: GRCz11 Supplementary files format and content: VST normalised expression values. Rows = genes; Columns = samples. Retina   Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   tissue:Retina|genotype:Choroideremia GSM8061006 GSM8061006: Choroideremia 5; Danio rerio; RNA Seq GSM8061006 r1 GSM8061006 1 Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP487850   loader:fastq load.py CHM5_2.fq.gz CHM5_1.fq.gz fastq fastq 17802156900.0 59340523.0 GSM8061006 r1 0:150 1:150 A:5198674613;C:3599354426;G:3698229931;T:5305840019;N:57911 150 150     5198674613 3599354426 3698229931 5305840019 57911 SRX23511745 SRS20362213 SRA1796957 The Francis Crick Institute The Francis Crick Institute 2 0.9109 0.91251 0.18901 0.18793 0.72354 0.73456 0.51839 0.52348 150 150 B B biological fallback assumption illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_plate smartseq   United Kingdom 2024-02-02 Undetermined Embryo Eye Sensory System
30582 30582 SRR27848823 SRX23511744 SRS20362212 SRP487850 PRJNA1072679 Oxidative stress altered glucose metabolism and inflammation contribute to the retinal phenotype in the choroideremia zebrafish GSE254948 Transcriptome Analysis Reactive oxygen species ROS within the retina play a key role in maintaining function and cell survival. However excessive ROS can lead to oxidative stress inducing dysregulation of metabolic and inflammatory pathways. The chmru848 zebrafish models choroideremia CHM an X linked chorioretinal dystrophy which predominantly affects the photoreceptors retinal pigment epithelium RPE and choroid. In this study we examined the transcriptomic signature of the chmru848 zebrafish retina to reveal upregulation of cytokine pathways and glia migration upregulation of oxidative ER stress and apoptosis markers and dysregulation of glucose metabolism with downregulation of glycolysis and upregulation of the oxidative phase of the pentose phosphate pathway. Glucose uptake was overall impaired in the chmru848 retina using the 2 NBDG glucose uptake assay but post overexpression of human PFKM it partially rescued the retinal phenotype and glucose uptake but without xxx the expression of glycolysis markers. Therapies targeting glucose metabolism in CHM may represent a potential remedial approach. Overall design: Choroideremia CHM is a x linked inherited retinal dystrophy caused by mutations in the CHM gene. It is a progressive condition leading to complete blindness. We are working on a chm zebrafish model which shows a widespread severe degenerative phenotype with embryos only surviving up to 5 days. We compared the transcriptome in wildtype vs chm zebrafish retinas and determine which pathways are disrupted in chm fish.       Choroideremia 4 GSM8061005   source name:Retina|tissue:Retina|genotype:Choroideremia|geo loc name:missing|collection date:missing Choroideremia 4 Raw reads were quality and adapter trimmed using trimgalore version 0.6.7 14 before alignment. Reads were mapped and subsequent gene level counted using RSEM 1.3.3 15 and STAR 2.7.10a 16 against the zebrafish genome GRCz11 both from Ensembl. Normalisation of raw count data and differential expression analysis was performed with the DESeq2 package version 1.38.3within the R programming environment version 4.2.2. Assembly: GRCz11 Supplementary files format and content: VST normalised expression values. Rows = genes; Columns = samples. Retina   Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   tissue:Retina|genotype:Choroideremia GSM8061005 GSM8061005: Choroideremia 4; Danio rerio; RNA Seq GSM8061005 r1 GSM8061005 1 Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP487850   loader:fastq load.py CHM4_2.fq.gz CHM4_1.fq.gz fastq fastq 15944831400.0 53149438.0 GSM8061005 r1 0:150 1:150 A:4833633886;C:2870981386;G:3055307522;T:5184819306;N:89300 150 150     4833633886 2870981386 3055307522 5184819306 89300 SRX23511744 SRS20362212 SRA1796957 The Francis Crick Institute The Francis Crick Institute 2 0.88929 0.88364 0.25277 0.24996 0.75317 0.77826 0.64914 0.65907 150 150 B B biological fallback assumption illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_plate smartseq   United Kingdom 2024-02-02 Undetermined Embryo Eye Sensory System
30583 30583 SRR27848824 SRX23511743 SRS20362211 SRP487850 PRJNA1072679 Oxidative stress altered glucose metabolism and inflammation contribute to the retinal phenotype in the choroideremia zebrafish GSE254948 Transcriptome Analysis Reactive oxygen species ROS within the retina play a key role in maintaining function and cell survival. However excessive ROS can lead to oxidative stress inducing dysregulation of metabolic and inflammatory pathways. The chmru848 zebrafish models choroideremia CHM an X linked chorioretinal dystrophy which predominantly affects the photoreceptors retinal pigment epithelium RPE and choroid. In this study we examined the transcriptomic signature of the chmru848 zebrafish retina to reveal upregulation of cytokine pathways and glia migration upregulation of oxidative ER stress and apoptosis markers and dysregulation of glucose metabolism with downregulation of glycolysis and upregulation of the oxidative phase of the pentose phosphate pathway. Glucose uptake was overall impaired in the chmru848 retina using the 2 NBDG glucose uptake assay but post overexpression of human PFKM it partially rescued the retinal phenotype and glucose uptake but without xxx the expression of glycolysis markers. Therapies targeting glucose metabolism in CHM may represent a potential remedial approach. Overall design: Choroideremia CHM is a x linked inherited retinal dystrophy caused by mutations in the CHM gene. It is a progressive condition leading to complete blindness. We are working on a chm zebrafish model which shows a widespread severe degenerative phenotype with embryos only surviving up to 5 days. We compared the transcriptome in wildtype vs chm zebrafish retinas and determine which pathways are disrupted in chm fish.       Choroideremia 3 GSM8061004   source name:Retina|tissue:Retina|genotype:Choroideremia|geo loc name:missing|collection date:missing Choroideremia 3 Raw reads were quality and adapter trimmed using trimgalore version 0.6.7 14 before alignment. Reads were mapped and subsequent gene level counted using RSEM 1.3.3 15 and STAR 2.7.10a 16 against the zebrafish genome GRCz11 both from Ensembl. Normalisation of raw count data and differential expression analysis was performed with the DESeq2 package version 1.38.3within the R programming environment version 4.2.2. Assembly: GRCz11 Supplementary files format and content: VST normalised expression values. Rows = genes; Columns = samples. Retina   Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   tissue:Retina|genotype:Choroideremia GSM8061004 GSM8061004: Choroideremia 3; Danio rerio; RNA Seq GSM8061004 r1 GSM8061004 1 Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP487850   loader:fastq load.py CHM3_1.fq.gz CHM3_2.fq.gz fastq fastq 16028608800.0 53428696.0 GSM8061004 r1 0:150 1:150 A:4842495383;C:2966448486;G:3084331699;T:5135033798;N:299434 150 150     4842495383 2966448486 3084331699 5135033798 299434 SRX23511743 SRS20362211 SRA1796957 The Francis Crick Institute The Francis Crick Institute 2 0.89747 0.88445 0.23706 0.23597 0.72904 0.75187 0.5514 0.45471 150 150 B B biological fallback assumption illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_plate smartseq   United Kingdom 2024-02-02 Undetermined Embryo Eye Sensory System
30584 30584 SRR27848825 SRX23511742 SRS20362210 SRP487850 PRJNA1072679 Oxidative stress altered glucose metabolism and inflammation contribute to the retinal phenotype in the choroideremia zebrafish GSE254948 Transcriptome Analysis Reactive oxygen species ROS within the retina play a key role in maintaining function and cell survival. However excessive ROS can lead to oxidative stress inducing dysregulation of metabolic and inflammatory pathways. The chmru848 zebrafish models choroideremia CHM an X linked chorioretinal dystrophy which predominantly affects the photoreceptors retinal pigment epithelium RPE and choroid. In this study we examined the transcriptomic signature of the chmru848 zebrafish retina to reveal upregulation of cytokine pathways and glia migration upregulation of oxidative ER stress and apoptosis markers and dysregulation of glucose metabolism with downregulation of glycolysis and upregulation of the oxidative phase of the pentose phosphate pathway. Glucose uptake was overall impaired in the chmru848 retina using the 2 NBDG glucose uptake assay but post overexpression of human PFKM it partially rescued the retinal phenotype and glucose uptake but without xxx the expression of glycolysis markers. Therapies targeting glucose metabolism in CHM may represent a potential remedial approach. Overall design: Choroideremia CHM is a x linked inherited retinal dystrophy caused by mutations in the CHM gene. It is a progressive condition leading to complete blindness. We are working on a chm zebrafish model which shows a widespread severe degenerative phenotype with embryos only surviving up to 5 days. We compared the transcriptome in wildtype vs chm zebrafish retinas and determine which pathways are disrupted in chm fish.       Choroideremia 2 GSM8061003   source name:Retina|tissue:Retina|genotype:Choroideremia|geo loc name:missing|collection date:missing Choroideremia 2 Raw reads were quality and adapter trimmed using trimgalore version 0.6.7 14 before alignment. Reads were mapped and subsequent gene level counted using RSEM 1.3.3 15 and STAR 2.7.10a 16 against the zebrafish genome GRCz11 both from Ensembl. Normalisation of raw count data and differential expression analysis was performed with the DESeq2 package version 1.38.3within the R programming environment version 4.2.2. Assembly: GRCz11 Supplementary files format and content: VST normalised expression values. Rows = genes; Columns = samples. Retina   Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   tissue:Retina|genotype:Choroideremia GSM8061003 GSM8061003: Choroideremia 2; Danio rerio; RNA Seq GSM8061003 r1 GSM8061003 1 Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP487850   loader:fastq load.py CHM2_1.fq.gz CHM2_2.fq.gz fastq fastq 14968884300.0 49896281.0 GSM8061003 r1 0:150 1:150 A:4575866591;C:2606132581;G:2819446784;T:4967389772;N:48572 150 150     4575866591 2606132581 2819446784 4967389772 48572 SRX23511742 SRS20362210 SRA1796957 The Francis Crick Institute The Francis Crick Institute 2 0.88022 0.87326 0.29312 0.29812 0.7335 0.76228 0.5854 0.57614 150 150 B B biological fallback assumption illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_plate smartseq   United Kingdom 2024-02-02 Undetermined Embryo Eye Sensory System
30585 30585 SRR27848826 SRX23511741 SRS20362209 SRP487850 PRJNA1072679 Oxidative stress altered glucose metabolism and inflammation contribute to the retinal phenotype in the choroideremia zebrafish GSE254948 Transcriptome Analysis Reactive oxygen species ROS within the retina play a key role in maintaining function and cell survival. However excessive ROS can lead to oxidative stress inducing dysregulation of metabolic and inflammatory pathways. The chmru848 zebrafish models choroideremia CHM an X linked chorioretinal dystrophy which predominantly affects the photoreceptors retinal pigment epithelium RPE and choroid. In this study we examined the transcriptomic signature of the chmru848 zebrafish retina to reveal upregulation of cytokine pathways and glia migration upregulation of oxidative ER stress and apoptosis markers and dysregulation of glucose metabolism with downregulation of glycolysis and upregulation of the oxidative phase of the pentose phosphate pathway. Glucose uptake was overall impaired in the chmru848 retina using the 2 NBDG glucose uptake assay but post overexpression of human PFKM it partially rescued the retinal phenotype and glucose uptake but without xxx the expression of glycolysis markers. Therapies targeting glucose metabolism in CHM may represent a potential remedial approach. Overall design: Choroideremia CHM is a x linked inherited retinal dystrophy caused by mutations in the CHM gene. It is a progressive condition leading to complete blindness. We are working on a chm zebrafish model which shows a widespread severe degenerative phenotype with embryos only surviving up to 5 days. We compared the transcriptome in wildtype vs chm zebrafish retinas and determine which pathways are disrupted in chm fish.       Choroideremia 1 GSM8061002   source name:Retina|tissue:Retina|genotype:Choroideremia|geo loc name:missing|collection date:missing Choroideremia 1 Raw reads were quality and adapter trimmed using trimgalore version 0.6.7 14 before alignment. Reads were mapped and subsequent gene level counted using RSEM 1.3.3 15 and STAR 2.7.10a 16 against the zebrafish genome GRCz11 both from Ensembl. Normalisation of raw count data and differential expression analysis was performed with the DESeq2 package version 1.38.3within the R programming environment version 4.2.2. Assembly: GRCz11 Supplementary files format and content: VST normalised expression values. Rows = genes; Columns = samples. Retina   Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   tissue:Retina|genotype:Choroideremia GSM8061002 GSM8061002: Choroideremia 1; Danio rerio; RNA Seq GSM8061002 r1 GSM8061002 1 Bulk RNA was extracted with RNAeasy Micro Plus kit Qiagen from chmru848 and wt control retinas n=7 per group and high RNA quality was confirmed by Agilent 2100 Bioanalyzer G2939A cDNA libraries were subsequently constructed from total RNA RIN ≥ 8 using the Clontech SMART Seq v4 Ultra Low Input RNA Kit   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP487850   loader:fastq load.py CHM1_2.fq.gz CHM1_1.fq.gz fastq fastq 15862639500.0 52875465.0 GSM8061002 r1 0:150 1:150 A:4812544148;C:2881706406;G:3014671171;T:5153419977;N:297798 150 150     4812544148 2881706406 3014671171 5153419977 297798 SRX23511741 SRS20362209 SRA1796957 The Francis Crick Institute The Francis Crick Institute 2 0.89157 0.87804 0.24616 0.24284 0.73884 0.76232 0.58251 0.58918 150 150 B B biological fallback assumption illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_plate smartseq   United Kingdom 2024-02-02 Undetermined Embryo Eye Sensory System
32194 32194 SRR29141332 SRX24663085 SRS21398383 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Multiome rgc:ntr preablation wildtype control day 5 RNA GSM8287442   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:no mtz|geo loc name:missing|collection date:missing Multiome rgc:ntr preablation wildtype control day 5 RNA scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:no mtz GSM8287442 GSM8287442: Multiome rgc:ntr preablation wildtype control day 5 RNA; Danio rerio; RNA Seq GSM8287442 r1 GSM8287442 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH182R_S1_L001_R1_001.fastq.gz TH182R_S1_L001_R2_001.fastq.gz fastq fastq 13807554173.0 116029867.0 GSM8287442 r1 0:28 1:91 A:4079582572;C:2862550576;G:2918085175;T:3947000701;N:335149 28 91     4079582572 2862550576 2918085175 3947000701 335149 SRX24663085 SRS21398383 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32195 32195 SRR29141333 SRX24663085 SRS21398383 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Multiome rgc:ntr preablation wildtype control day 5 RNA GSM8287442   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:no mtz|geo loc name:missing|collection date:missing Multiome rgc:ntr preablation wildtype control day 5 RNA scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:no mtz GSM8287442 GSM8287442: Multiome rgc:ntr preablation wildtype control day 5 RNA; Danio rerio; RNA Seq GSM8287442 r1 GSM8287442 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH182R_S1_L002_R1_001.fastq.gz TH182R_S1_L002_R2_001.fastq.gz fastq fastq 13803126064.0 115992656.0 GSM8287442 r2 0:28 1:91 A:4078989982;C:2861718905;G:2916062814;T:3946005912;N:348451 28 91     4078989982 2861718905 2916062814 3946005912 348451 SRX24663085 SRS21398383 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32196 32196 SRR29141334 SRX24663085 SRS21398383 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Multiome rgc:ntr preablation wildtype control day 5 RNA GSM8287442   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:no mtz|geo loc name:missing|collection date:missing Multiome rgc:ntr preablation wildtype control day 5 RNA scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:no mtz GSM8287442 GSM8287442: Multiome rgc:ntr preablation wildtype control day 5 RNA; Danio rerio; RNA Seq GSM8287442 r1 GSM8287442 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH182R_S1_L003_R1_001.fastq.gz TH182R_S1_L003_R2_001.fastq.gz fastq fastq 14039364150.0 117977850.0 GSM8287442 r3 0:28 1:91 A:4148421178;C:2910249087;G:2967774612;T:4012572976;N:346297 28 91     4148421178 2910249087 2967774612 4012572976 346297 SRX24663085 SRS21398383 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32197 32197 SRR29141335 SRX24663085 SRS21398383 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Multiome rgc:ntr preablation wildtype control day 5 RNA GSM8287442   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:no mtz|geo loc name:missing|collection date:missing Multiome rgc:ntr preablation wildtype control day 5 RNA scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:no mtz GSM8287442 GSM8287442: Multiome rgc:ntr preablation wildtype control day 5 RNA; Danio rerio; RNA Seq GSM8287442 r1 GSM8287442 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH182R_S1_L004_R1_001.fastq.gz TH182R_S1_L004_R2_001.fastq.gz fastq fastq 14396508973.0 120979067.0 GSM8287442 r4 0:28 1:91 A:4252694309;C:2985039203;G:3045994377;T:4112440375;N:340709 28 91     4252694309 2985039203 3045994377 4112440375 340709 SRX24663085 SRS21398383 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32198 32198 SRR29141336 SRX24663084 SRS21398382 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 7 24h mtz GSM8287437   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 7 24h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287437 GSM8287437: Ablated rgc:ntr day 7 24h mtz; Danio rerio; RNA Seq GSM8287437 r1 GSM8287437 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH111_S65_R1_001.fastq.gz TH111_S65_R2_001.fastq.gz fastq fastq 10009743191.0 84115489.0 GSM8287437 r1 0:28 1:91 A:2899067167;C:2137099335;G:2274433670;T:2671820531;N:27322488 28 91     2899067167 2137099335 2274433670 2671820531 27322488 SRX24663084 SRS21398382 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32199 32199 SRR29141337 SRX24663084 SRS21398382 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 7 24h mtz GSM8287437   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 7 24h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287437 GSM8287437: Ablated rgc:ntr day 7 24h mtz; Danio rerio; RNA Seq GSM8287437 r1 GSM8287437 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH111_S66_R1_001.fastq.gz TH111_S66_R2_001.fastq.gz fastq fastq 10310944686.0 86646594.0 GSM8287437 r2 0:28 1:91 A:2991162064;C:2200127755;G:2335919653;T:2755298440;N:28436774 28 91     2991162064 2200127755 2335919653 2755298440 28436774 SRX24663084 SRS21398382 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32200 32200 SRR29141338 SRX24663084 SRS21398382 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 7 24h mtz GSM8287437   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 7 24h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287437 GSM8287437: Ablated rgc:ntr day 7 24h mtz; Danio rerio; RNA Seq GSM8287437 r1 GSM8287437 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH111_S68_R1_001.fastq.gz TH111_S68_R2_001.fastq.gz fastq fastq 13987726480.0 117543920.0 GSM8287437 r3 0:28 1:91 A:4045259735;C:2989556292;G:3181238633;T:3733480210;N:38191610 28 91     4045259735 2989556292 3181238633 3733480210 38191610 SRX24663084 SRS21398382 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32201 32201 SRR29141339 SRX24663084 SRS21398382 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 7 24h mtz GSM8287437   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 7 24h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287437 GSM8287437: Ablated rgc:ntr day 7 24h mtz; Danio rerio; RNA Seq GSM8287437 r1 GSM8287437 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH111_S67_R1_001.fastq.gz TH111_S67_R2_001.fastq.gz fastq fastq 11876682069.0 99804051.0 GSM8287437 r4 0:28 1:91 A:3432787756;C:2538691346;G:2701757248;T:3170892876;N:32552843 28 91     3432787756 2538691346 2701757248 3170892876 32552843 SRX24663084 SRS21398382 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32202 32202 SRR29141340 SRX24663083 SRS21398381 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 9 72h mtz GSM8287441   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 9 72h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287441 GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq GSM8287441 r1 GSM8287441 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH136_S61_L001_R1_001.fastq.gz TH136_S61_L001_R2_001.fastq.gz fastq fastq 4774895706.0 40125174.0 GSM8287441 r1 0:28 1:91 A:1351265716;C:1042777184;G:1100567875;T:1280161557;N:123374 28 91     1351265716 1042777184 1100567875 1280161557 123374 SRX24663083 SRS21398381 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32203 32203 SRR29141341 SRX24663083 SRS21398381 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 9 72h mtz GSM8287441   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 9 72h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287441 GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq GSM8287441 r1 GSM8287441 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH136_S64_L003_R1_001.fastq.gz TH136_S64_L003_R2_001.fastq.gz fastq fastq 5538657579.0 46543341.0 GSM8287441 r10 0:28 1:91 A:1567217187;C:1211616483;G:1275241958;T:1484449584;N:132367 28 91     1567217187 1211616483 1275241958 1484449584 132367 SRX24663083 SRS21398381 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32204 32204 SRR29141342 SRX24663083 SRS21398381 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 9 72h mtz GSM8287441   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 9 72h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287441 GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq GSM8287441 r1 GSM8287441 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH136_S64_L004_R1_001.fastq.gz TH136_S64_L004_R2_001.fastq.gz fastq fastq 5403469057.0 45407303.0 GSM8287441 r11 0:28 1:91 A:1530355512;C:1180686287;G:1242924943;T:1449389493;N:112822 28 91     1530355512 1180686287 1242924943 1449389493 112822 SRX24663083 SRS21398381 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32205 32205 SRR29141343 SRX24663083 SRS21398381 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 9 72h mtz GSM8287441   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 9 72h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287441 GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq GSM8287441 r1 GSM8287441 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH136_S61_L002_R1_001.fastq.gz TH136_S61_L002_R2_001.fastq.gz fastq fastq 4786338627.0 40221333.0 GSM8287441 r12 0:28 1:91 A:1355122219;C:1044516973;G:1101760300;T:1284824360;N:114775 28 91     1355122219 1044516973 1101760300 1284824360 114775 SRX24663083 SRS21398381 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32206 32206 SRR29141344 SRX24663083 SRS21398381 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 9 72h mtz GSM8287441   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 9 72h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287441 GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq GSM8287441 r1 GSM8287441 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH136_S62_L001_R1_001.fastq.gz TH136_S62_L001_R2_001.fastq.gz fastq fastq 4901760535.0 41191265.0 GSM8287441 r13 0:28 1:91 A:1391714409;C:1068852387;G:1124767182;T:1316299153;N:127404 28 91     1391714409 1068852387 1124767182 1316299153 127404 SRX24663083 SRS21398381 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32207 32207 SRR29141345 SRX24663083 SRS21398381 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 9 72h mtz GSM8287441   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 9 72h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287441 GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq GSM8287441 r1 GSM8287441 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH136_S62_L004_R1_001.fastq.gz TH136_S62_L004_R2_001.fastq.gz fastq fastq 4937865968.0 41494672.0 GSM8287441 r14 0:28 1:91 A:1399404530;C:1078622301;G:1134489765;T:1325245845;N:103527 28 91     1399404530 1078622301 1134489765 1325245845 103527 SRX24663083 SRS21398381 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32208 32208 SRR29141346 SRX24663083 SRS21398381 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 9 72h mtz GSM8287441   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 9 72h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287441 GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq GSM8287441 r1 GSM8287441 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH136_S63_L003_R1_001.fastq.gz TH136_S63_L003_R2_001.fastq.gz fastq fastq 4497873940.0 37797260.0 GSM8287441 r15 0:28 1:91 A:1271859460;C:985043807;G:1034974096;T:1205889617;N:106960 28 91     1271859460 985043807 1034974096 1205889617 106960 SRX24663083 SRS21398381 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32209 32209 SRR29141347 SRX24663083 SRS21398381 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 9 72h mtz GSM8287441   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 9 72h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287441 GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq GSM8287441 r1 GSM8287441 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH136_S64_L002_R1_001.fastq.gz TH136_S64_L002_R2_001.fastq.gz fastq fastq 5236234787.0 44001973.0 GSM8287441 r16 0:28 1:91 A:1486231119;C:1141729846;G:1201608482;T:1406539690;N:125650 28 91     1486231119 1141729846 1201608482 1406539690 125650 SRX24663083 SRS21398381 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32210 32210 SRR29141348 SRX24663083 SRS21398381 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 9 72h mtz GSM8287441   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 9 72h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287441 GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq GSM8287441 r1 GSM8287441 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH136_S61_L003_R1_001.fastq.gz TH136_S61_L003_R2_001.fastq.gz fastq fastq 5047989877.0 42420083.0 GSM8287441 r2 0:28 1:91 A:1424658452;C:1105197562;G:1165585488;T:1352428003;N:120372 28 91     1424658452 1105197562 1165585488 1352428003 120372 SRX24663083 SRS21398381 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32211 32211 SRR29141349 SRX24663083 SRS21398381 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 9 72h mtz GSM8287441   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 9 72h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287441 GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq GSM8287441 r1 GSM8287441 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH136_S61_L004_R1_001.fastq.gz TH136_S61_L004_R2_001.fastq.gz fastq fastq 4922634682.0 41366678.0 GSM8287441 r3 0:28 1:91 A:1390880945;C:1076385249;G:1135254796;T:1320011695;N:101997 28 91     1390880945 1076385249 1135254796 1320011695 101997 SRX24663083 SRS21398381 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32212 32212 SRR29141350 SRX24663083 SRS21398381 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 9 72h mtz GSM8287441   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 9 72h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287441 GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq GSM8287441 r1 GSM8287441 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH136_S62_L002_R1_001.fastq.gz TH136_S62_L002_R2_001.fastq.gz fastq fastq 4903434746.0 41205334.0 GSM8287441 r4 0:28 1:91 A:1392700449;C:1068492247;G:1123677701;T:1318447188;N:117161 28 91     1392700449 1068492247 1123677701 1318447188 117161 SRX24663083 SRS21398381 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32213 32213 SRR29141351 SRX24663083 SRS21398381 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 9 72h mtz GSM8287441   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 9 72h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287441 GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq GSM8287441 r1 GSM8287441 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH136_S62_L003_R1_001.fastq.gz TH136_S62_L003_R2_001.fastq.gz fastq fastq 5079175136.0 42682144.0 GSM8287441 r5 0:28 1:91 A:1438032514;C:1110731492;G:1168141629;T:1362147196;N:122305 28 91     1438032514 1110731492 1168141629 1362147196 122305 SRX24663083 SRS21398381 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32214 32214 SRR29141352 SRX24663083 SRS21398381 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 9 72h mtz GSM8287441   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 9 72h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287441 GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq GSM8287441 r1 GSM8287441 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH136_S63_L001_R1_001.fastq.gz TH136_S63_L001_R2_001.fastq.gz fastq fastq 4248222293.0 35699347.0 GSM8287441 r6 0:28 1:91 A:1204282194;C:928219300;G:975617558;T:1139994829;N:108412 28 91     1204282194 928219300 975617558 1139994829 108412 SRX24663083 SRS21398381 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32215 32215 SRR29141353 SRX24663083 SRS21398381 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 9 72h mtz GSM8287441   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 9 72h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287441 GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq GSM8287441 r1 GSM8287441 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH136_S63_L002_R1_001.fastq.gz TH136_S63_L002_R2_001.fastq.gz fastq fastq 4256614411.0 35769869.0 GSM8287441 r7 0:28 1:91 A:1207165748;C:929375627;G:976155057;T:1143817351;N:100628 28 91     1207165748 929375627 976155057 1143817351 100628 SRX24663083 SRS21398381 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32216 32216 SRR29141354 SRX24663083 SRS21398381 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 9 72h mtz GSM8287441   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 9 72h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287441 GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq GSM8287441 r1 GSM8287441 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH136_S63_L004_R1_001.fastq.gz TH136_S63_L004_R2_001.fastq.gz fastq fastq 4376437534.0 36776786.0 GSM8287441 r8 0:28 1:91 A:1238798346;C:957263182;G:1005958070;T:1174327377;N:90559 28 91     1238798346 957263182 1005958070 1174327377 90559 SRX24663083 SRS21398381 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32217 32217 SRR29141355 SRX24663083 SRS21398381 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 9 72h mtz GSM8287441   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 9 72h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287441 GSM8287441: Ablated rgc:ntr day 9 72h mtz; Danio rerio; RNA Seq GSM8287441 r1 GSM8287441 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH136_S64_L001_R1_001.fastq.gz TH136_S64_L001_R2_001.fastq.gz fastq fastq 5222497546.0 43886534.0 GSM8287441 r9 0:28 1:91 A:1481856778;C:1139695420;G:1200133908;T:1400676121;N:135319 28 91     1481856778 1139695420 1200133908 1400676121 135319 SRX24663083 SRS21398381 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32218 32218 SRR29141356 SRX24663082 SRS21398380 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 6 12h mtz GSM8287440   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 6 12h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287440 GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq GSM8287440 r1 GSM8287440 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH135_S57_L001_R1_001.fastq.gz TH135_S57_L001_R2_001.fastq.gz fastq fastq 4775761788.0 40132452.0 GSM8287440 r1 0:28 1:91 A:1364997508;C:1030536901;G:1087120578;T:1292982564;N:124237 28 91     1364997508 1030536901 1087120578 1292982564 124237 SRX24663082 SRS21398380 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32219 32219 SRR29141357 SRX24663082 SRS21398380 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 6 12h mtz GSM8287440   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 6 12h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287440 GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq GSM8287440 r1 GSM8287440 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH135_S60_L002_R1_001.fastq.gz TH135_S60_L002_R2_001.fastq.gz fastq fastq 5081243356.0 42699524.0 GSM8287440 r10 0:28 1:91 A:1453763884;C:1095418883;G:1154387721;T:1377550690;N:122178 28 91     1453763884 1095418883 1154387721 1377550690 122178 SRX24663082 SRS21398380 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32220 32220 SRR29141358 SRX24663082 SRS21398380 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 6 12h mtz GSM8287440   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 6 12h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287440 GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq GSM8287440 r1 GSM8287440 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH135_S60_L004_R1_001.fastq.gz TH135_S60_L004_R2_001.fastq.gz fastq fastq 5258119482.0 44185878.0 GSM8287440 r11 0:28 1:91 A:1501504527;C:1135881534;G:1197368112;T:1423254847;N:110462 28 91     1501504527 1135881534 1197368112 1423254847 110462 SRX24663082 SRS21398380 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32221 32221 SRR29141359 SRX24663082 SRS21398380 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 6 12h mtz GSM8287440   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 6 12h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287440 GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq GSM8287440 r1 GSM8287440 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH135_S57_L003_R1_001.fastq.gz TH135_S57_L003_R2_001.fastq.gz fastq fastq 5008964184.0 42092136.0 GSM8287440 r12 0:28 1:91 A:1428212274;C:1083563490;G:1142704874;T:1354362571;N:120975 28 91     1428212274 1083563490 1142704874 1354362571 120975 SRX24663082 SRS21398380 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32222 32222 SRR29141360 SRX24663082 SRS21398380 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 6 12h mtz GSM8287440   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 6 12h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287440 GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq GSM8287440 r1 GSM8287440 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH135_S58_L002_R1_001.fastq.gz TH135_S58_L002_R2_001.fastq.gz fastq fastq 5035503207.0 42315153.0 GSM8287440 r13 0:28 1:91 A:1440077950;C:1086334843;G:1144377517;T:1364592460;N:120437 28 91     1440077950 1086334843 1144377517 1364592460 120437 SRX24663082 SRS21398380 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32223 32223 SRR29141361 SRX24663082 SRS21398380 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 6 12h mtz GSM8287440   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 6 12h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287440 GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq GSM8287440 r1 GSM8287440 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH135_S59_L001_R1_001.fastq.gz TH135_S59_L001_R2_001.fastq.gz fastq fastq 5387099536.0 45269744.0 GSM8287440 r14 0:28 1:91 A:1537849058;C:1163912984;G:1227124886;T:1458073621;N:138987 28 91     1537849058 1163912984 1227124886 1458073621 138987 SRX24663082 SRS21398380 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32224 32224 SRR29141362 SRX24663082 SRS21398380 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 6 12h mtz GSM8287440   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 6 12h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287440 GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq GSM8287440 r1 GSM8287440 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH135_S59_L004_R1_001.fastq.gz TH135_S59_L004_R2_001.fastq.gz fastq fastq 5512521490.0 46323710.0 GSM8287440 r15 0:28 1:91 A:1571984524;C:1192238932;G:1256792838;T:1491389888;N:115308 28 91     1571984524 1192238932 1256792838 1491389888 115308 SRX24663082 SRS21398380 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32225 32225 SRR29141363 SRX24663082 SRS21398380 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 6 12h mtz GSM8287440   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 6 12h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287440 GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq GSM8287440 r1 GSM8287440 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH135_S60_L003_R1_001.fastq.gz TH135_S60_L003_R2_001.fastq.gz fastq fastq 5376169386.0 45177894.0 GSM8287440 r16 0:28 1:91 A:1533658425;C:1162739111;G:1225658907;T:1453983443;N:129500 28 91     1533658425 1162739111 1225658907 1453983443 129500 SRX24663082 SRS21398380 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32226 32226 SRR29141364 SRX24663082 SRS21398380 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 6 12h mtz GSM8287440   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 6 12h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287440 GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq GSM8287440 r1 GSM8287440 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH135_S57_L002_R1_001.fastq.gz TH135_S57_L002_R2_001.fastq.gz fastq fastq 4787380115.0 40230085.0 GSM8287440 r2 0:28 1:91 A:1368967190;C:1032261634;G:1088378612;T:1297658513;N:114166 28 91     1368967190 1032261634 1088378612 1297658513 114166 SRX24663082 SRS21398380 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32227 32227 SRR29141365 SRX24663082 SRS21398380 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 6 12h mtz GSM8287440   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 6 12h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287440 GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq GSM8287440 r1 GSM8287440 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH135_S57_L004_R1_001.fastq.gz TH135_S57_L004_R2_001.fastq.gz fastq fastq 4885123145.0 41051455.0 GSM8287440 r3 0:28 1:91 A:1394450278;C:1055564129;G:1113114189;T:1321891090;N:103459 28 91     1394450278 1055564129 1113114189 1321891090 103459 SRX24663082 SRS21398380 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32228 32228 SRR29141366 SRX24663082 SRS21398380 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 6 12h mtz GSM8287440   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 6 12h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287440 GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq GSM8287440 r1 GSM8287440 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH135_S58_L001_R1_001.fastq.gz TH135_S58_L001_R2_001.fastq.gz fastq fastq 5060757982.0 42527378.0 GSM8287440 r4 0:28 1:91 A:1446313544;C:1092878434;G:1151738503;T:1369695242;N:132259 28 91     1446313544 1092878434 1151738503 1369695242 132259 SRX24663082 SRS21398380 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32229 32229 SRR29141367 SRX24663082 SRS21398380 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 6 12h mtz GSM8287440   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 6 12h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287440 GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq GSM8287440 r1 GSM8287440 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH135_S58_L003_R1_001.fastq.gz TH135_S58_L003_R2_001.fastq.gz fastq fastq 5322898560.0 44730240.0 GSM8287440 r5 0:28 1:91 A:1517934750;C:1151967941;G:1213786668;T:1439081677;N:127524 28 91     1517934750 1151967941 1213786668 1439081677 127524 SRX24663082 SRS21398380 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32230 32230 SRR29141368 SRX24663082 SRS21398380 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 6 12h mtz GSM8287440   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 6 12h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287440 GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq GSM8287440 r1 GSM8287440 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH135_S58_L004_R1_001.fastq.gz TH135_S58_L004_R2_001.fastq.gz fastq fastq 5160850786.0 43368494.0 GSM8287440 r6 0:28 1:91 A:1473328102;C:1115516878;G:1175502159;T:1396395641;N:108006 28 91     1473328102 1115516878 1175502159 1396395641 108006 SRX24663082 SRS21398380 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32231 32231 SRR29141369 SRX24663082 SRS21398380 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 6 12h mtz GSM8287440   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 6 12h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287440 GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq GSM8287440 r1 GSM8287440 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH135_S59_L002_R1_001.fastq.gz TH135_S59_L002_R2_001.fastq.gz fastq fastq 5381504513.0 45222727.0 GSM8287440 r7 0:28 1:91 A:1537238062;C:1161721381;G:1224135102;T:1458280892;N:129076 28 91     1537238062 1161721381 1224135102 1458280892 129076 SRX24663082 SRS21398380 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32232 32232 SRR29141370 SRX24663082 SRS21398380 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 6 12h mtz GSM8287440   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 6 12h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287440 GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq GSM8287440 r1 GSM8287440 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH135_S59_L003_R1_001.fastq.gz TH135_S59_L003_R2_001.fastq.gz fastq fastq 5665367827.0 47608133.0 GSM8287440 r8 0:28 1:91 A:1613897652;C:1226812698;G:1293000098;T:1531521187;N:136192 28 91     1613897652 1226812698 1293000098 1531521187 136192 SRX24663082 SRS21398380 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32233 32233 SRR29141371 SRX24663082 SRS21398380 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Ablated rgc:ntr day 6 12h mtz GSM8287440   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:mtz|geo loc name:missing|collection date:missing Ablated rgc:ntr day 6 12h mtz scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:mtz GSM8287440 GSM8287440: Ablated rgc:ntr day 6 12h mtz; Danio rerio; RNA Seq GSM8287440 r1 GSM8287440 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH135_S60_L001_R1_001.fastq.gz TH135_S60_L001_R2_001.fastq.gz fastq fastq 5045550377.0 42399583.0 GSM8287440 r9 0:28 1:91 A:1442885146;C:1088672739;G:1147630180;T:1366230521;N:131791 28 91     1442885146 1088672739 1147630180 1366230521 131791 SRX24663082 SRS21398380 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32234 32234 SRR29141372 SRX24663081 SRS21398379 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Unablated rgc:ntr day 9 GSM8287439   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:no mtz|geo loc name:missing|collection date:missing Unablated rgc:ntr day 9 scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:no mtz GSM8287439 GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq GSM8287439 r1 GSM8287439 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH134_S53_L001_R1_001.fastq.gz TH134_S53_L001_R2_001.fastq.gz fastq fastq 4566870378.0 38377062.0 GSM8287439 r1 0:28 1:91 A:1300193484;C:990777876;G:1047244494;T:1228536400;N:118124 28 91     1300193484 990777876 1047244494 1228536400 118124 SRX24663081 SRS21398379 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32235 32235 SRR29141373 SRX24663081 SRS21398379 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Unablated rgc:ntr day 9 GSM8287439   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:no mtz|geo loc name:missing|collection date:missing Unablated rgc:ntr day 9 scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:no mtz GSM8287439 GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq GSM8287439 r1 GSM8287439 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH134_S55_L002_R1_001.fastq.gz TH134_S55_L002_R2_001.fastq.gz fastq fastq 4872575309.0 40946011.0 GSM8287439 r10 0:28 1:91 A:1388373514;C:1056698672;G:1113330484;T:1314056163;N:116476 28 91     1388373514 1056698672 1113330484 1314056163 116476 SRX24663081 SRS21398379 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32236 32236 SRR29141374 SRX24663081 SRS21398379 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Unablated rgc:ntr day 9 GSM8287439   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:no mtz|geo loc name:missing|collection date:missing Unablated rgc:ntr day 9 scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:no mtz GSM8287439 GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq GSM8287439 r1 GSM8287439 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH134_S55_L003_R1_001.fastq.gz TH134_S55_L003_R2_001.fastq.gz fastq fastq 5138052528.0 43176912.0 GSM8287439 r11 0:28 1:91 A:1459926271;C:1117597394;G:1177808023;T:1382597761;N:123079 28 91     1459926271 1117597394 1177808023 1382597761 123079 SRX24663081 SRS21398379 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32237 32237 SRR29141375 SRX24663081 SRS21398379 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Unablated rgc:ntr day 9 GSM8287439   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:no mtz|geo loc name:missing|collection date:missing Unablated rgc:ntr day 9 scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:no mtz GSM8287439 GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq GSM8287439 r1 GSM8287439 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH134_S55_L004_R1_001.fastq.gz TH134_S55_L004_R2_001.fastq.gz fastq fastq 5022868263.0 42208977.0 GSM8287439 r12 0:28 1:91 A:1428914798;C:1091192534;G:1150147876;T:1352508063;N:104992 28 91     1428914798 1091192534 1150147876 1352508063 104992 SRX24663081 SRS21398379 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32238 32238 SRR29141376 SRX24663081 SRS21398379 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Unablated rgc:ntr day 9 GSM8287439   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:no mtz|geo loc name:missing|collection date:missing Unablated rgc:ntr day 9 scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:no mtz GSM8287439 GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq GSM8287439 r1 GSM8287439 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH134_S56_L001_R1_001.fastq.gz TH134_S56_L001_R2_001.fastq.gz fastq fastq 4569776953.0 38401487.0 GSM8287439 r13 0:28 1:91 A:1299842950;C:992232187;G:1048131188;T:1229452715;N:117913 28 91     1299842950 992232187 1048131188 1229452715 117913 SRX24663081 SRS21398379 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32239 32239 SRR29141377 SRX24663081 SRS21398379 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Unablated rgc:ntr day 9 GSM8287439   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:no mtz|geo loc name:missing|collection date:missing Unablated rgc:ntr day 9 scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:no mtz GSM8287439 GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq GSM8287439 r1 GSM8287439 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH134_S56_L002_R1_001.fastq.gz TH134_S56_L002_R2_001.fastq.gz fastq fastq 4604427135.0 38692665.0 GSM8287439 r14 0:28 1:91 A:1310481305;C:998823194;G:1054488359;T:1240523819;N:110458 28 91     1310481305 998823194 1054488359 1240523819 110458 SRX24663081 SRS21398379 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval Eye Sensory System
32240 32240 SRR29141378 SRX24663081 SRS21398379 SRP509393 PRJNA1115053 Large scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific GSE268179 Other Many genes are known to regulate Müller glia MG dependent retinal regeneration following widespread tissue damage. Conversely genes controlling regeneration following limited retinal cell loss per degenerative disease are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here transgenic zebrafish enabling inducible selective retinal ganglion cell RGC ablation were combined with single cell multiomics and CRISPR/Cas9 based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration and seven of these knockouts actually enhanced RGC replacement kinetics including sox2 olig2 and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e. biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem like state and context specificity in how genes function during regeneration. Increased understanding of how disease relevant cell types can be selectively regenerated will support the development of disease tailored regenerative therapeutics. Overall design: We performed single cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells RGCs 4 timepoints and ablation of rod photoreceptors and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.   pubmed:39007397   Unablated rgc:ntr day 9 GSM8287439   source name:Eye|tissue:Eye|transgenic line:rgc:ntr|treatment:no mtz|geo loc name:missing|collection date:missing Unablated rgc:ntr day 9 scRNAseq: Raw reads were mapped to the Danio rerio GRCz10 using Cell Ranger v7.0 from 10x genomics. Aligned genomic reads were then read into the published Seurat pipeline v4.3.0.1 and quality control was performed by removing any cells with <200 detected genes or 1000 UMIs and genes detected in fewer than 3 cells per experiment. Clustering steps were performed using steps from the pbmc Seurat tutorial available online. Briefly the top 2 000 variable genes were identified and used to identify principal components PCs of the data. The top 30 PCs were used to produce a UMAP and clusters were annotated with known zebrafish marker genes. Differentially expressed genes DEGs were identified using the FindAllMarkers function between each control and ablation timepoint in each retinal cell cluster minimum log2 foldchange cutoff of 0.25. scMultiomeseq: RNA expression data was processed as above. Peak calling from single nuclei ATAC seq reads was performed using MACS2 in the ArchR package v1.0.2. ATAC seq data was then processed using the pbmc scATAC seq workflow with the Signac v1.10.0 and Seurat v4.3.0.1 packages for quality control normalization and producing an integrated UMAP. Differential expression and accessibility was then calculated for both gene RNA expression and chromatin peak accessibility. Next the ChromVar package v1.18.0 was used to identify differentially accessible transcription factor motifs between wildtype and ascl1a mutant cells. Assembly: GRCz11 Supplementary files format and content: Cellular expression data varies in format either as h5 standalone files or barcodes features and matrix files to be used together. ATAC data is available as standalone fragment.tsv files Eye   scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   tissue:Eye|transgenic line:rgc:ntr|treatment:no mtz GSM8287439 GSM8287439: Unablated rgc:ntr day 9; Danio rerio; RNA Seq GSM8287439 r1 GSM8287439 1 scRNAseq: 40 60 eyes were dissected from sibling fish and subsequently placed in 20 U/ml papain 10 eyes per 1 ml Worthington and incubated at 28°C for 30 min with gentle agitation. Cells were pelleted and resuspended in PBS containing 0.1 mg/ml leupeptin Sigma Aldrich and 10 U/ml DNaseI Roche. Cells were filtered through a 70 μm filter Miltenyi Biotec kept on ice until 10X genomics processing. scMultiomeseq: 40 60 eyes were dissected and flash frozen in dry ice for 15min before being transferred to a 80 C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression GEX protocol CGOOO338. Briefly frozen retinal tissues were lysed in ice cold 500ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of 3000 5000 nuclei/ml and kept on ice until 10x genomics processing. Library preparation was then performed according to 10x genomics protocols.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP509393   loader:fastq load.py TH134_S56_L003_R1_001.fastq.gz TH134_S56_L003_R2_001.fastq.gz fastq fastq 4894210580.0 41127820.0 GSM8287439 r15 0:28 1:91 A:1389263901;C:1064650503;G:1124176385;T:1316003245;N:116546 28 91     1389263901 1064650503 1124176385 1316003245 116546 SRX24663081 SRS21398379 SRA1875751 Jeff Mumm, Ophthalmology, Johns Hopkins University Jeff Mumm, Ophthalmology, Johns Hopkins University                       T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2024-05-23 Undetermined Larval 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");;