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run_metadata

839 rows where experiment.library_layout = "PAIRED", experiment.library_selection = "cDNA" and tissue_curation = "Eye"

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devstage_curation 8

technology 7

devstage_curation_coarse 6

experiment.platform 2

experiment.library_source 2

tissue_curation_coarse 1

tissue_curation 1

  • Eye · 839 ✖

experiment.library_strategy 1

experiment.library_selection 1

  • cDNA · 839 ✖

experiment.library_layout 1

  • PAIRED · 839 ✖
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
25123 25123 SRR25605440 SRX21332624 SRS18578256 SRP454539 PRJNA1004255 Unique activities of two overlapping PAX6 retinal enhancers GSE240575 Transcriptome Analysis Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.   pubmed:37643867   gfp enriched rep1 GSM7702831   source name:Eye|tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf|geo loc name:missing|collection date:missing gfp enriched rep1 Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted… Eye   NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.   tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf GSM7702831 GSM7702831: gfp enriched rep1; Danio rerio; RNA Seq GSM7702831 r1 GSM7702831 1 NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA NextSeq 2000   SRP454539   loader:fastq load.py gfp_nre_S1_L001_R2_001.fastq.gz gfp_nre_S1_L001_R1_001.fastq.gz gfp_nre_S1_L001_I2_001.fastq.gz gfp_nre_S1_L001_I1_001.fastq.gz fastq fastq fastq fastq 47873499684.0 346909418.0 GSM7702831 r1 0:10 1:10 2:28 3:90 A:8912593789;C:6786299583;G:7771099171;T:7737013838;N:14841239 10 10 28 90 8912593789 6786299583 7771099171 7737013838 14841239 SRX21332624 SRS18578256 SRA1690580 Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh 1 0.90717   0.15854   0.80501   0.50927   90   B   usable mapping rate illumina nextseq_v2 3prime cdna_unspecified unknown sc single_cell_droplet 10x   United Kingdom 2023-08-10 Hatching Embryo Eye Sensory System
25124 25124 SRR25605441 SRX21332624 SRS18578256 SRP454539 PRJNA1004255 Unique activities of two overlapping PAX6 retinal enhancers GSE240575 Transcriptome Analysis Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.   pubmed:37643867   gfp enriched rep1 GSM7702831   source name:Eye|tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf|geo loc name:missing|collection date:missing gfp enriched rep1 Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted… Eye   NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.   tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf GSM7702831 GSM7702831: gfp enriched rep1; Danio rerio; RNA Seq GSM7702831 r1 GSM7702831 1 NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA NextSeq 2000   SRP454539   loader:fastq load.py gfp_nre_S1_L002_I1_001.fastq.gz gfp_nre_S1_L002_I2_001.fastq.gz gfp_nre_S1_L002_R1_001.fastq.gz gfp_nre_S1_L002_R2_001.fastq.gz fastq fastq fastq fastq 48659347860.0 352603970.0 GSM7702831 r2 0:10 1:10 2:28 3:90 A:9056524463;C:6888387179;G:7922807308;T:7854042793;N:12595557 10 10 28 90 9056524463 6888387179 7922807308 7854042793 12595557 SRX21332624 SRS18578256 SRA1690580 Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh 1 0.90541   0.15686   0.80543   0.51051   90   B   usable mapping rate illumina nextseq_v2 3prime cdna_unspecified unknown sc single_cell_droplet 10x   United Kingdom 2023-08-10 Hatching Embryo Eye Sensory System
25125 25125 SRR25605442 SRX21332623 SRS18578255 SRP454539 PRJNA1004255 Unique activities of two overlapping PAX6 retinal enhancers GSE240575 Transcriptome Analysis Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.   pubmed:37643867   mcherry enriched rep1 GSM7702830   source name:Eye|tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf|geo loc name:missing|collection date:missing mcherry enriched rep1 Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted… Eye   NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.   tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf GSM7702830 GSM7702830: mcherry enriched rep1; Danio rerio; RNA Seq GSM7702830 r1 GSM7702830 1 NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA NextSeq 2000   SRP454539   loader:fastq load.py double_pos_S2_L001_R2_001.fastq.gz double_pos_S2_L001_R1_001.fastq.gz double_pos_S2_L001_I2_001.fastq.gz double_pos_S2_L001_I1_001.fastq.gz fastq fastq fastq fastq 41371188084.0 299791218.0 GSM7702830 r1 0:10 1:10 2:28 3:90 A:7690531012;C:5787287634;G:6514950407;T:6975618202;N:12822365 10 10 28 90 7690531012 5787287634 6514950407 6975618202 12822365 SRX21332623 SRS18578255 SRA1690580 Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh 1 0.93894   0.17677   0.79926   0.51122   90   B   usable mapping rate illumina nextseq_v2 3prime cdna_unspecified unknown sc single_cell_droplet 10x   United Kingdom 2023-08-10 Hatching Embryo Eye Sensory System
25126 25126 SRR25605443 SRX21332623 SRS18578255 SRP454539 PRJNA1004255 Unique activities of two overlapping PAX6 retinal enhancers GSE240575 Transcriptome Analysis Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.   pubmed:37643867   mcherry enriched rep1 GSM7702830   source name:Eye|tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf|geo loc name:missing|collection date:missing mcherry enriched rep1 Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted… Eye   NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.   tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf GSM7702830 GSM7702830: mcherry enriched rep1; Danio rerio; RNA Seq GSM7702830 r1 GSM7702830 1 NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA NextSeq 2000   SRP454539   loader:fastq load.py double_pos_S2_L002_I1_001.fastq.gz double_pos_S2_L002_I2_001.fastq.gz double_pos_S2_L002_R1_001.fastq.gz double_pos_S2_L002_R2_001.fastq.gz fastq fastq fastq fastq 42692812980.0 309368210.0 GSM7702830 r2 0:10 1:10 2:28 3:90 A:7933061913;C:5964617781;G:6744802923;T:7189579846;N:11076437 10 10 28 90 7933061913 5964617781 6744802923 7189579846 11076437 SRX21332623 SRS18578255 SRA1690580 Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh 1 0.93887   0.17578   0.79825   0.50169   90   B   usable mapping rate illumina nextseq_v2 3prime cdna_unspecified unknown sc single_cell_droplet 10x   United Kingdom 2023-08-10 Hatching 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
29107 29107 SRR27151543 SRX22833143 SRS19813583 SRP476691 PRJNA1050282 Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf whole eye] GSE249751 Transcriptome Analysis The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 / retina red cones are transformed to hybrid red/UV sensitive cones green cones are transfated to blue cones and the number of rods is greatly reduced. In the mouse Samd7 / retina dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf larvae samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. parent bioproject:PRJNA1050288 pubmed:39531499   5 dpf larval eyes samd7 / rep3 GSM7963641   source name:eye|tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / |geo loc name:missing|collection date:missing 5 dpf larval eyes samd7 / rep3 Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression DESeq2 1.34.0 was used in R 4.1.3 using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 / adult retina Table S3 we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 / and WT replicates derived from 5dpf larval eyes. Genes are ranked by p adj. eye   To perform RNA seq on 5 dpf larvae samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / GSM7963641 GSM7963641: 5 dpf larval eyes samd7 / rep3; Danio rerio; RNA Seq GSM7963641 r1 GSM7963641 1 To perform RNA seq on 5 dpf larvae samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP476691   loader:fastq load.py k7.AGGTATTCGG-GAGTCGCTTC.H37F5DSX2_AGGTATTCGG-GAGTCGCTTC_L003_R1.fastq.gz k7.AGGTATTCGG-GAGTCGCTTC.H37F5DSX2_AGGTATTCGG-GAGTCGCTTC_L003_R2.fastq.gz fastq fastq 10747100652.0 35586426.0 GSM7963641 r1 0:151 1:151 A:2879574125;C:2414500329;G:2693618198;T:2759237631;N:170369 151 151     2879574125 2414500329 2693618198 2759237631 170369 SRX22833143 SRS19813583 SRA1765718 Pathology and Immunology, Washington University School of Medicine Pathology and Immunology, Washington University School of Medicine 2 0.95486 0.95989 0.05038 0.05167 0.73018 0.73438 0.41476 0.42743 151 151 B B biological fallback assumption illumina novaseq_era 3prime random_priming smarter bulk unknown unknown   United States 2023-12-08 Larval Larval Eye Sensory System
29108 29108 SRR27151544 SRX22833142 SRS19813582 SRP476691 PRJNA1050282 Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf whole eye] GSE249751 Transcriptome Analysis The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 / retina red cones are transformed to hybrid red/UV sensitive cones green cones are transfated to blue cones and the number of rods is greatly reduced. In the mouse Samd7 / retina dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf larvae samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. parent bioproject:PRJNA1050288 pubmed:39531499   5 dpf larval eyes samd7 / rep2 GSM7963640   source name:eye|tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / |geo loc name:missing|collection date:missing 5 dpf larval eyes samd7 / rep2 Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression DESeq2 1.34.0 was used in R 4.1.3 using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 / adult retina Table S3 we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 / and WT replicates derived from 5dpf larval eyes. Genes are ranked by p adj. eye   To perform RNA seq on 5 dpf larvae samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / GSM7963640 GSM7963640: 5 dpf larval eyes samd7 / rep2; Danio rerio; RNA Seq GSM7963640 r1 GSM7963640 1 To perform RNA seq on 5 dpf larvae samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP476691   loader:fastq load.py k10.AATCGAACTC-TTAGACCTTC.H37F5DSX2_AATCGAACTC-TTAGACCTTC_L003_R1.fastq.gz k10.AATCGAACTC-TTAGACCTTC.H37F5DSX2_AATCGAACTC-TTAGACCTTC_L003_R2.fastq.gz fastq fastq 10739126946.0 35560023.0 GSM7963640 r1 0:151 1:151 A:2888527520;C:2418475992;G:2670968816;T:2760983764;N:170854 151 151     2888527520 2418475992 2670968816 2760983764 170854 SRX22833142 SRS19813582 SRA1765718 Pathology and Immunology, Washington University School of Medicine Pathology and Immunology, Washington University School of Medicine 2 0.95306 0.95846 0.05658 0.05746 0.73269 0.73714 0.42191 0.43088 151 151 B B biological fallback assumption illumina novaseq_era 3prime random_priming smarter bulk unknown unknown   United States 2023-12-08 Larval Larval Eye Sensory System
29109 29109 SRR27151545 SRX22833141 SRS19813581 SRP476691 PRJNA1050282 Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf whole eye] GSE249751 Transcriptome Analysis The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 / retina red cones are transformed to hybrid red/UV sensitive cones green cones are transfated to blue cones and the number of rods is greatly reduced. In the mouse Samd7 / retina dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf larvae samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. parent bioproject:PRJNA1050288 pubmed:39531499   5 dpf larval eyes samd7 / rep1 GSM7963639   source name:eye|tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / |geo loc name:missing|collection date:missing 5 dpf larval eyes samd7 / rep1 Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression DESeq2 1.34.0 was used in R 4.1.3 using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 / adult retina Table S3 we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 / and WT replicates derived from 5dpf larval eyes. Genes are ranked by p adj. eye   To perform RNA seq on 5 dpf larvae samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / GSM7963639 GSM7963639: 5 dpf larval eyes samd7 / rep1; Danio rerio; RNA Seq GSM7963639 r1 GSM7963639 1 To perform RNA seq on 5 dpf larvae samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP476691   loader:fastq load.py k11.CCGAAGATAA-TGGATTAACG.H37F5DSX2_CCGAAGATAA-TGGATTAACG_L003_R1.fastq.gz k11.CCGAAGATAA-TGGATTAACG.H37F5DSX2_CCGAAGATAA-TGGATTAACG_L003_R2.fastq.gz fastq fastq 10514289154.0 34815527.0 GSM7963639 r1 0:151 1:151 A:2862247233;C:2377266250;G:2528917605;T:2745692826;N:165240 151 151     2862247233 2377266250 2528917605 2745692826 165240 SRX22833141 SRS19813581 SRA1765718 Pathology and Immunology, Washington University School of Medicine Pathology and Immunology, Washington University School of Medicine 2 0.95376 0.95966 0.05526 0.05681 0.72965 0.73273 0.42276 0.43148 151 151 B B biological fallback assumption illumina novaseq_era 3prime random_priming smarter bulk unknown unknown   United States 2023-12-08 Larval Larval Eye Sensory System
29110 29110 SRR27151546 SRX22833140 SRS19813578 SRP476691 PRJNA1050282 Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf whole eye] GSE249751 Transcriptome Analysis The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 / retina red cones are transformed to hybrid red/UV sensitive cones green cones are transfated to blue cones and the number of rods is greatly reduced. In the mouse Samd7 / retina dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf larvae samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. parent bioproject:PRJNA1050288 pubmed:39531499   5 dpf larval eyes WT rep3 GSM7963638   source name:eye|tissue:eye|developmental stage:5 dpf larvae|genotype:WT|geo loc name:missing|collection date:missing 5 dpf larval eyes WT rep3 Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression DESeq2 1.34.0 was used in R 4.1.3 using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 / adult retina Table S3 we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 / and WT replicates derived from 5dpf larval eyes. Genes are ranked by p adj. eye   To perform RNA seq on 5 dpf larvae samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   tissue:eye|developmental stage:5 dpf larvae|genotype:WT GSM7963638 GSM7963638: 5 dpf larval eyes WT rep3; Danio rerio; RNA Seq GSM7963638 r1 GSM7963638 1 To perform RNA seq on 5 dpf larvae samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP476691   loader:fastq load.py w6.CCTTGCCGTA-TAGTACACAG.H37F5DSX2_CCTTGCCGTA-TAGTACACAG_L003_R1.fastq.gz w6.CCTTGCCGTA-TAGTACACAG.H37F5DSX2_CCTTGCCGTA-TAGTACACAG_L003_R2.fastq.gz fastq fastq 11943994904.0 39549652.0 GSM7963638 r1 0:151 1:151 A:3182349655;C:2708994785;G:2976982793;T:3075478468;N:189203 151 151     3182349655 2708994785 2976982793 3075478468 189203 SRX22833140 SRS19813578 SRA1765718 Pathology and Immunology, Washington University School of Medicine Pathology and Immunology, Washington University School of Medicine 2 0.95522 0.96109 0.04859 0.04955 0.74247 0.74799 0.42424 0.43226 151 151 B B biological fallback assumption illumina novaseq_era 3prime random_priming smarter bulk unknown unknown   United States 2023-12-08 Larval Larval Eye Sensory System
29111 29111 SRR27151547 SRX22833139 SRS19813579 SRP476691 PRJNA1050282 Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf whole eye] GSE249751 Transcriptome Analysis The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 / retina red cones are transformed to hybrid red/UV sensitive cones green cones are transfated to blue cones and the number of rods is greatly reduced. In the mouse Samd7 / retina dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf larvae samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. parent bioproject:PRJNA1050288 pubmed:39531499   5 dpf larval eyes WT rep2 GSM7963637   source name:eye|tissue:eye|developmental stage:5 dpf larvae|genotype:WT|geo loc name:missing|collection date:missing 5 dpf larval eyes WT rep2 Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression DESeq2 1.34.0 was used in R 4.1.3 using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 / adult retina Table S3 we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 / and WT replicates derived from 5dpf larval eyes. Genes are ranked by p adj. eye   To perform RNA seq on 5 dpf larvae samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   tissue:eye|developmental stage:5 dpf larvae|genotype:WT GSM7963637 GSM7963637: 5 dpf larval eyes WT rep2; Danio rerio; RNA Seq GSM7963637 r1 GSM7963637 1 To perform RNA seq on 5 dpf larvae samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP476691   loader:fastq load.py w8.AATCTCAGGC-TCACCAGGAC.H37F5DSX2_AATCTCAGGC-TCACCAGGAC_L003_R1.fastq.gz w8.AATCTCAGGC-TCACCAGGAC.H37F5DSX2_AATCTCAGGC-TCACCAGGAC_L003_R2.fastq.gz fastq fastq 10471048190.0 34672345.0 GSM7963637 r1 0:151 1:151 A:2767212261;C:2402799397;G:2644524852;T:2656347723;N:163957 151 151     2767212261 2402799397 2644524852 2656347723 163957 SRX22833139 SRS19813579 SRA1765718 Pathology and Immunology, Washington University School of Medicine Pathology and Immunology, Washington University School of Medicine 2 0.95909 0.96443 0.04132 0.04289 0.74217 0.74631 0.40379 0.41076 151 151 B B biological fallback assumption illumina novaseq_era 3prime random_priming smarter bulk unknown unknown   United States 2023-12-08 Larval Larval Eye Sensory System
29112 29112 SRR27151548 SRX22833138 SRS19813580 SRP476691 PRJNA1050282 Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf whole eye] GSE249751 Transcriptome Analysis The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 / retina red cones are transformed to hybrid red/UV sensitive cones green cones are transfated to blue cones and the number of rods is greatly reduced. In the mouse Samd7 / retina dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf larvae samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. parent bioproject:PRJNA1050288 pubmed:39531499   5 dpf larval eyes WT rep1 GSM7963636   source name:eye|tissue:eye|developmental stage:5 dpf larvae|genotype:WT|geo loc name:missing|collection date:missing 5 dpf larval eyes WT rep1 Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression DESeq2 1.34.0 was used in R 4.1.3 using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 / adult retina Table S3 we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 / and WT replicates derived from 5dpf larval eyes. Genes are ranked by p adj. eye   To perform RNA seq on 5 dpf larvae samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   tissue:eye|developmental stage:5 dpf larvae|genotype:WT GSM7963636 GSM7963636: 5 dpf larval eyes WT rep1; Danio rerio; RNA Seq GSM7963636 r1 GSM7963636 1 To perform RNA seq on 5 dpf larvae samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring. Larvae were euthanized by exposure to tricaine anterior halves of the body were collected in RNAlater Invitrogen and stored at 4°C and corresponding posterior halves were collected in 50 mM NaOH for genotyping. Once genotypes were confirmed eyes were dissected from the anterior body halves. The eyes from an individual larva constituted one replicate. Three WT and samd7stl888/stl888 replicates were collected and RNA extracted using the RNeasy Micro Kit Qiagen then DNAse treated and repurified using the Rneasy MinElute Cleanup Kit Qiagen. RNA concentrations ranged from 1.5 2.2 ng/µl 20 ng RNA total with prominent 18/28S peaks and minimal RNA degradation in Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP476691   loader:fastq load.py w15.AACAGGCAAG-TTCATGGAAG.H37F5DSX2_AACAGGCAAG-TTCATGGAAG_L003_R1.fastq.gz w15.AACAGGCAAG-TTCATGGAAG.H37F5DSX2_AACAGGCAAG-TTCATGGAAG_L003_R2.fastq.gz fastq fastq 11852131940.0 39245470.0 GSM7963636 r1 0:151 1:151 A:3157263060;C:2691924686;G:2998627934;T:3004129760;N:186500 151 151     3157263060 2691924686 2998627934 3004129760 186500 SRX22833138 SRS19813580 SRA1765718 Pathology and Immunology, Washington University School of Medicine Pathology and Immunology, Washington University School of Medicine 2 0.95652 0.96043 0.05012 0.05113 0.73198 0.73637 0.42361 0.41636 151 151 B B biological fallback assumption illumina novaseq_era 3prime random_priming smarter bulk unknown unknown   United States 2023-12-08 Larval Larval Eye Sensory System
29113 29113 SRR27151348 SRX22832950 SRS19813390 SRP476686 PRJNA1050286 Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [adult] GSE249754 Transcriptome Analysis The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 / retina red cones are transformed to hybrid red/UV sensitive cones green cones are transfated to blue cones and the number of rods is greatly reduced. In the mouse Samd7 / retina dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 3 mpf adult retinas samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring and grown to maturity. post adults were genotyped pairs of retinas from three male WT and samd7stl888/stl888 zebrafish were dissected. The pair of retinas from an individual zebrafish were then combined to make one replicate and RNA was extracted using the RNEasy Mini Kit Qiagen with on column DNAse treatment using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 20 37 ng/µl 600 1100 ng total with RIN scores ranging from 9 9.5; there was minimal RNA degradation as shown by Bioanalyzer traces. parent bioproject:PRJNA1050288 pubmed:39531499   Adult retina samd7 / rep3 GSM7963654   source name:retina|tissue:retina|developmental stage:adult|genotype:samd7 / |geo loc name:missing|collection date:missing Adult retina samd7 / rep3 Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression DESeq2 1.34.0 was used in R 4.1.3 using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 / adult retina Table S3 we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 / and WT replicates derived from adult retina. Genes are ranked by p adj. retina   To perform RNA seq on 3 mpf adult retinas samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring and grown to maturity. post adults were genotyped pairs of retinas from three male WT and samd7stl888/stl888 zebrafish were dissected. The pair of retinas from an individual zebrafish were then combined to make one replicate and RNA was extracted using the RNEasy Mini Kit Qiagen with on column DNAse treatment using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 20 37 ng/µl 600 1100 ng total with RIN scores ranging from 9 9.5; there was minimal RNA degradation as shown by Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   tissue:retina|developmental stage:adult|genotype:samd7 / GSM7963654 GSM7963654: Adult retina samd7 / rep3; Danio rerio; RNA Seq GSM7963654 r1 GSM7963654 1 To perform RNA seq on 3 mpf adult retinas samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring and grown to maturity. post adults were genotyped pairs of retinas from three male WT and samd7stl888/stl888 zebrafish were dissected. The pair of retinas from an individual zebrafish were then combined to make one replicate and RNA was extracted using the RNEasy Mini Kit Qiagen with on column DNAse treatment using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 20 37 ng/µl 600 1100 ng total with RIN scores ranging from 9 9.5; there was minimal RNA degradation as shown by Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP476686   loader:fastq load.py samd7 3.GTCGACTCCT-ACCTTAGCCG.HJNJTDSX3_GTCGACTCCT-ACCTTAGCCG_L003_R2.fastq.gz samd7 3.GTCGACTCCT-ACCTTAGCCG.HJNJTDSX3_GTCGACTCCT-ACCTTAGCCG_L003_R1.fastq.gz fastq fastq 12350621596.0 40896098.0 GSM7963654 r1 0:151 1:151 A:3311452403;C:2805529783;G:3049468848;T:3184114382;N:56180 151 151     3311452403 2805529783 3049468848 3184114382 56180 SRX22832950 SRS19813390 SRA1765771 Pathology and Immunology, Washington University School of Medicine Pathology and Immunology, Washington University School of Medicine 2 0.9517 0.9527 0.08772 0.08879 0.73342 0.73574 0.50684 0.51229 151 151 B B biological fallback assumption illumina novaseq_era 3prime cdna_unspecified smarter bulk unknown unknown   United States 2023-12-08 Adult Adult Eye Sensory System
29114 29114 SRR27151349 SRX22832949 SRS19813389 SRP476686 PRJNA1050286 Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [adult] GSE249754 Transcriptome Analysis The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 / retina red cones are transformed to hybrid red/UV sensitive cones green cones are transfated to blue cones and the number of rods is greatly reduced. In the mouse Samd7 / retina dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 3 mpf adult retinas samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring and grown to maturity. post adults were genotyped pairs of retinas from three male WT and samd7stl888/stl888 zebrafish were dissected. The pair of retinas from an individual zebrafish were then combined to make one replicate and RNA was extracted using the RNEasy Mini Kit Qiagen with on column DNAse treatment using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 20 37 ng/µl 600 1100 ng total with RIN scores ranging from 9 9.5; there was minimal RNA degradation as shown by Bioanalyzer traces. parent bioproject:PRJNA1050288 pubmed:39531499   Adult retina samd7 / rep2 GSM7963653   source name:retina|tissue:retina|developmental stage:adult|genotype:samd7 / |geo loc name:missing|collection date:missing Adult retina samd7 / rep2 Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression DESeq2 1.34.0 was used in R 4.1.3 using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 / adult retina Table S3 we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 / and WT replicates derived from adult retina. Genes are ranked by p adj. retina   To perform RNA seq on 3 mpf adult retinas samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring and grown to maturity. post adults were genotyped pairs of retinas from three male WT and samd7stl888/stl888 zebrafish were dissected. The pair of retinas from an individual zebrafish were then combined to make one replicate and RNA was extracted using the RNEasy Mini Kit Qiagen with on column DNAse treatment using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 20 37 ng/µl 600 1100 ng total with RIN scores ranging from 9 9.5; there was minimal RNA degradation as shown by Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   tissue:retina|developmental stage:adult|genotype:samd7 / GSM7963653 GSM7963653: Adult retina samd7 / rep2; Danio rerio; RNA Seq GSM7963653 r1 GSM7963653 1 To perform RNA seq on 3 mpf adult retinas samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring and grown to maturity. post adults were genotyped pairs of retinas from three male WT and samd7stl888/stl888 zebrafish were dissected. The pair of retinas from an individual zebrafish were then combined to make one replicate and RNA was extracted using the RNEasy Mini Kit Qiagen with on column DNAse treatment using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 20 37 ng/µl 600 1100 ng total with RIN scores ranging from 9 9.5; there was minimal RNA degradation as shown by Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP476686   loader:fastq load.py samd7 2.AACACCAATG-GCACTTACAA.HJNJTDSX3_AACACCAATG-GCACTTACAA_L003_R2.fastq.gz samd7 2.AACACCAATG-GCACTTACAA.HJNJTDSX3_AACACCAATG-GCACTTACAA_L003_R1.fastq.gz fastq fastq 10779711820.0 35694410.0 GSM7963653 r1 0:151 1:151 A:2944031330;C:2416631581;G:2607925305;T:2811076060;N:47544 151 151     2944031330 2416631581 2607925305 2811076060 47544 SRX22832949 SRS19813389 SRA1765771 Pathology and Immunology, Washington University School of Medicine Pathology and Immunology, Washington University School of Medicine 2 0.94587 0.94807 0.0994 0.10023 0.73837 0.74052 0.50262 0.50849 151 151 B B biological fallback assumption illumina novaseq_era 3prime cdna_unspecified smarter bulk unknown unknown   United States 2023-12-08 Adult Adult Eye Sensory System
29115 29115 SRR27151350 SRX22832948 SRS19813387 SRP476686 PRJNA1050286 Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [adult] GSE249754 Transcriptome Analysis The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 / retina red cones are transformed to hybrid red/UV sensitive cones green cones are transfated to blue cones and the number of rods is greatly reduced. In the mouse Samd7 / retina dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 3 mpf adult retinas samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring and grown to maturity. post adults were genotyped pairs of retinas from three male WT and samd7stl888/stl888 zebrafish were dissected. The pair of retinas from an individual zebrafish were then combined to make one replicate and RNA was extracted using the RNEasy Mini Kit Qiagen with on column DNAse treatment using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 20 37 ng/µl 600 1100 ng total with RIN scores ranging from 9 9.5; there was minimal RNA degradation as shown by Bioanalyzer traces. parent bioproject:PRJNA1050288 pubmed:39531499   Adult retina samd7 / rep1 GSM7963652   source name:retina|tissue:retina|developmental stage:adult|genotype:samd7 / |geo loc name:missing|collection date:missing Adult retina samd7 / rep1 Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression DESeq2 1.34.0 was used in R 4.1.3 using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 / adult retina Table S3 we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 / and WT replicates derived from adult retina. Genes are ranked by p adj. retina   To perform RNA seq on 3 mpf adult retinas samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring and grown to maturity. post adults were genotyped pairs of retinas from three male WT and samd7stl888/stl888 zebrafish were dissected. The pair of retinas from an individual zebrafish were then combined to make one replicate and RNA was extracted using the RNEasy Mini Kit Qiagen with on column DNAse treatment using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 20 37 ng/µl 600 1100 ng total with RIN scores ranging from 9 9.5; there was minimal RNA degradation as shown by Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   tissue:retina|developmental stage:adult|genotype:samd7 / GSM7963652 GSM7963652: Adult retina samd7 / rep1; Danio rerio; RNA Seq GSM7963652 r1 GSM7963652 1 To perform RNA seq on 3 mpf adult retinas samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring and grown to maturity. post adults were genotyped pairs of retinas from three male WT and samd7stl888/stl888 zebrafish were dissected. The pair of retinas from an individual zebrafish were then combined to make one replicate and RNA was extracted using the RNEasy Mini Kit Qiagen with on column DNAse treatment using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 20 37 ng/µl 600 1100 ng total with RIN scores ranging from 9 9.5; there was minimal RNA degradation as shown by Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP476686   loader:fastq load.py samd7 1.AGAACTGGTG-TGGATTGTAG.HJNJTDSX3_AGAACTGGTG-TGGATTGTAG_L003_R1.fastq.gz samd7 1.AGAACTGGTG-TGGATTGTAG.HJNJTDSX3_AGAACTGGTG-TGGATTGTAG_L003_R2.fastq.gz fastq fastq 10592879520.0 35075760.0 GSM7963652 r1 0:151 1:151 A:2902239961;C:2373635807;G:2559549595;T:2757407466;N:46691 151 151     2902239961 2373635807 2559549595 2757407466 46691 SRX22832948 SRS19813387 SRA1765771 Pathology and Immunology, Washington University School of Medicine Pathology and Immunology, Washington University School of Medicine 2 0.94718 0.94948 0.09134 0.09292 0.7291 0.73117 0.51985 0.52005 151 151 B B biological fallback assumption illumina novaseq_era 3prime cdna_unspecified smarter bulk unknown unknown   United States 2023-12-08 Adult Adult Eye Sensory System
29116 29116 SRR27151351 SRX22832947 SRS19813388 SRP476686 PRJNA1050286 Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [adult] GSE249754 Transcriptome Analysis The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 / retina red cones are transformed to hybrid red/UV sensitive cones green cones are transfated to blue cones and the number of rods is greatly reduced. In the mouse Samd7 / retina dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 3 mpf adult retinas samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring and grown to maturity. post adults were genotyped pairs of retinas from three male WT and samd7stl888/stl888 zebrafish were dissected. The pair of retinas from an individual zebrafish were then combined to make one replicate and RNA was extracted using the RNEasy Mini Kit Qiagen with on column DNAse treatment using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 20 37 ng/µl 600 1100 ng total with RIN scores ranging from 9 9.5; there was minimal RNA degradation as shown by Bioanalyzer traces. parent bioproject:PRJNA1050288 pubmed:39531499   Adult retina WT rep3 GSM7963651   source name:retina|tissue:retina|developmental stage:adult|genotype:WT|geo loc name:missing|collection date:missing Adult retina WT rep3 Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression DESeq2 1.34.0 was used in R 4.1.3 using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 / adult retina Table S3 we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 / and WT replicates derived from adult retina. Genes are ranked by p adj. retina   To perform RNA seq on 3 mpf adult retinas samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring and grown to maturity. post adults were genotyped pairs of retinas from three male WT and samd7stl888/stl888 zebrafish were dissected. The pair of retinas from an individual zebrafish were then combined to make one replicate and RNA was extracted using the RNEasy Mini Kit Qiagen with on column DNAse treatment using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 20 37 ng/µl 600 1100 ng total with RIN scores ranging from 9 9.5; there was minimal RNA degradation as shown by Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   tissue:retina|developmental stage:adult|genotype:WT GSM7963651 GSM7963651: Adult retina WT rep3; Danio rerio; RNA Seq GSM7963651 r1 GSM7963651 1 To perform RNA seq on 3 mpf adult retinas samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring and grown to maturity. post adults were genotyped pairs of retinas from three male WT and samd7stl888/stl888 zebrafish were dissected. The pair of retinas from an individual zebrafish were then combined to make one replicate and RNA was extracted using the RNEasy Mini Kit Qiagen with on column DNAse treatment using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 20 37 ng/µl 600 1100 ng total with RIN scores ranging from 9 9.5; there was minimal RNA degradation as shown by Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP476686   loader:fastq load.py wt 3.TCCGAAGTGG-ACACAATGGT.HJNJTDSX3_TCCGAAGTGG-ACACAATGGT_L003_R1.fastq.gz wt 3.TCCGAAGTGG-ACACAATGGT.HJNJTDSX3_TCCGAAGTGG-ACACAATGGT_L003_R2.fastq.gz fastq fastq 11864600916.0 39286758.0 GSM7963651 r1 0:151 1:151 A:3134961848;C:2656797227;G:3067645293;T:3005143714;N:52834 151 151     3134961848 2656797227 3067645293 3005143714 52834 SRX22832947 SRS19813388 SRA1765771 Pathology and Immunology, Washington University School of Medicine Pathology and Immunology, Washington University School of Medicine 2 0.94961 0.95129 0.08731 0.08788 0.75465 0.75739 0.52752 0.51933 151 151 B B biological fallback assumption illumina novaseq_era 3prime cdna_unspecified smarter bulk unknown unknown   United States 2023-12-08 Adult Adult Eye Sensory System
29117 29117 SRR27151352 SRX22832946 SRS19813385 SRP476686 PRJNA1050286 Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [adult] GSE249754 Transcriptome Analysis The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 / retina red cones are transformed to hybrid red/UV sensitive cones green cones are transfated to blue cones and the number of rods is greatly reduced. In the mouse Samd7 / retina dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 3 mpf adult retinas samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring and grown to maturity. post adults were genotyped pairs of retinas from three male WT and samd7stl888/stl888 zebrafish were dissected. The pair of retinas from an individual zebrafish were then combined to make one replicate and RNA was extracted using the RNEasy Mini Kit Qiagen with on column DNAse treatment using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 20 37 ng/µl 600 1100 ng total with RIN scores ranging from 9 9.5; there was minimal RNA degradation as shown by Bioanalyzer traces. parent bioproject:PRJNA1050288 pubmed:39531499   Adult retina WT rep2 GSM7963650   source name:retina|tissue:retina|developmental stage:adult|genotype:WT|geo loc name:missing|collection date:missing Adult retina WT rep2 Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression DESeq2 1.34.0 was used in R 4.1.3 using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 / adult retina Table S3 we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 / and WT replicates derived from adult retina. Genes are ranked by p adj. retina   To perform RNA seq on 3 mpf adult retinas samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring and grown to maturity. post adults were genotyped pairs of retinas from three male WT and samd7stl888/stl888 zebrafish were dissected. The pair of retinas from an individual zebrafish were then combined to make one replicate and RNA was extracted using the RNEasy Mini Kit Qiagen with on column DNAse treatment using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 20 37 ng/µl 600 1100 ng total with RIN scores ranging from 9 9.5; there was minimal RNA degradation as shown by Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   tissue:retina|developmental stage:adult|genotype:WT GSM7963650 GSM7963650: Adult retina WT rep2; Danio rerio; RNA Seq GSM7963650 r1 GSM7963650 1 To perform RNA seq on 3 mpf adult retinas samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring and grown to maturity. post adults were genotyped pairs of retinas from three male WT and samd7stl888/stl888 zebrafish were dissected. The pair of retinas from an individual zebrafish were then combined to make one replicate and RNA was extracted using the RNEasy Mini Kit Qiagen with on column DNAse treatment using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 20 37 ng/µl 600 1100 ng total with RIN scores ranging from 9 9.5; there was minimal RNA degradation as shown by Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP476686   loader:fastq load.py wt 2.CGATAGCAGG-TAAGTGTCGA.HJNJTDSX3_CGATAGCAGG-TAAGTGTCGA_L003_R1.fastq.gz wt 2.CGATAGCAGG-TAAGTGTCGA.HJNJTDSX3_CGATAGCAGG-TAAGTGTCGA_L003_R2.fastq.gz fastq fastq 11537286672.0 38202936.0 GSM7963650 r1 0:151 1:151 A:3128579183;C:2619829672;G:2797531802;T:2991294275;N:51740 151 151     3128579183 2619829672 2797531802 2991294275 51740 SRX22832946 SRS19813385 SRA1765771 Pathology and Immunology, Washington University School of Medicine Pathology and Immunology, Washington University School of Medicine 2 0.95108 0.9523 0.08612 0.08648 0.754 0.75643 0.5265 0.51457 151 151 B B biological fallback assumption illumina novaseq_era 3prime cdna_unspecified smarter bulk unknown unknown   United States 2023-12-08 Adult Adult Eye Sensory System
29118 29118 SRR27151353 SRX22832945 SRS19813386 SRP476686 PRJNA1050286 Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [adult] GSE249754 Transcriptome Analysis The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 / retina red cones are transformed to hybrid red/UV sensitive cones green cones are transfated to blue cones and the number of rods is greatly reduced. In the mouse Samd7 / retina dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 3 mpf adult retinas samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring and grown to maturity. post adults were genotyped pairs of retinas from three male WT and samd7stl888/stl888 zebrafish were dissected. The pair of retinas from an individual zebrafish were then combined to make one replicate and RNA was extracted using the RNEasy Mini Kit Qiagen with on column DNAse treatment using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 20 37 ng/µl 600 1100 ng total with RIN scores ranging from 9 9.5; there was minimal RNA degradation as shown by Bioanalyzer traces. parent bioproject:PRJNA1050288 pubmed:39531499   Adult retina WT rep1 GSM7963649   source name:retina|tissue:retina|developmental stage:adult|genotype:WT|geo loc name:missing|collection date:missing Adult retina WT rep1 Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression DESeq2 1.34.0 was used in R 4.1.3 using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 / adult retina Table S3 we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 / and WT replicates derived from adult retina. Genes are ranked by p adj. retina   To perform RNA seq on 3 mpf adult retinas samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring and grown to maturity. post adults were genotyped pairs of retinas from three male WT and samd7stl888/stl888 zebrafish were dissected. The pair of retinas from an individual zebrafish were then combined to make one replicate and RNA was extracted using the RNEasy Mini Kit Qiagen with on column DNAse treatment using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 20 37 ng/µl 600 1100 ng total with RIN scores ranging from 9 9.5; there was minimal RNA degradation as shown by Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   tissue:retina|developmental stage:adult|genotype:WT GSM7963649 GSM7963649: Adult retina WT rep1; Danio rerio; RNA Seq GSM7963649 r1 GSM7963649 1 To perform RNA seq on 3 mpf adult retinas samd7stl888/+ heterozygous were intercrossed to produce mixed genotype offspring and grown to maturity. post adults were genotyped pairs of retinas from three male WT and samd7stl888/stl888 zebrafish were dissected. The pair of retinas from an individual zebrafish were then combined to make one replicate and RNA was extracted using the RNEasy Mini Kit Qiagen with on column DNAse treatment using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 20 37 ng/µl 600 1100 ng total with RIN scores ranging from 9 9.5; there was minimal RNA degradation as shown by Bioanalyzer traces. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP476686   loader:fastq load.py wt 1.AACACCGGTT-ACGAGACGTC.HJNJTDSX3_AACACCGGTT-ACGAGACGTC_L003_R2.fastq.gz wt 1.AACACCGGTT-ACGAGACGTC.HJNJTDSX3_AACACCGGTT-ACGAGACGTC_L003_R1.fastq.gz fastq fastq 15142192118.0 50139709.0 GSM7963649 r1 0:151 1:151 A:4123269688;C:3413024919;G:3665579705;T:3940250217;N:67589 151 151     4123269688 3413024919 3665579705 3940250217 67589 SRX22832945 SRS19813386 SRA1765771 Pathology and Immunology, Washington University School of Medicine Pathology and Immunology, Washington University School of Medicine 2 0.94752 0.94913 0.08393 0.08515 0.75446 0.75672 0.46043 0.506 151 151 B B biological fallback assumption illumina novaseq_era 3prime cdna_unspecified smarter bulk unknown unknown   United States 2023-12-08 Adult Adult Eye Sensory System
29119 29119 SRR27151354 SRX22832956 SRS19813396 SRP476687 PRJNA1050291 Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf td] GSE249755 Transcriptome Analysis The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 / retina red cones are transformed to hybrid red/UV sensitive cones green cones are transfated to blue cones and the number of rods is greatly reduced. In the mouse Samd7 / retina dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf thrb:tdTomato+ cells the following crosses were performed: WT × WT;thrb:tdTomato+/ or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS as described in another section. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total with RNA integrity RIN scores ranging from 7.4 8.4. parent bioproject:PRJNA1050288 pubmed:39531499   thrb:tdTomato+ cells from 5 dpf larval eyes samd7 / rep3 GSM7963660   source name:eye|cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / |geo loc name:missing|collection date:missing thrb:tdTomato+ cells from 5 dpf larval eyes samd7 / rep3 Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression DESeq2 1.34.0 was used in R 4.1.3 using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 / adult retina Table S3 we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 / and WT replicates derived from 5dpf thrb:tdTomato+ cells. Genes are ranked by p adj. eye   To perform RNA seq on 5 dpf thrb:tdTomato+ cells the following crosses were performed: WT × WT;thrb:tdTomato+/ or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / GSM7963660 GSM7963660: thrb:tdTomato+ cells from 5 dpf larval eyes samd7 / rep3; Danio rerio; RNA Seq GSM7963660 r1 GSM7963660 1 To perform RNA seq on 5 dpf thrb:tdTomato+ cells the following crosses were performed: WT × WT;thrb:tdTomato+/ or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP476687   loader:fastq load.py samd7_1_rna_6_2_22.AGAAGGAACA-TGGTAGATGC.HNGJJDSX3_AGAAGGAACA-TGGTAGATGC_L003_R1.fastq.gz samd7_1_rna_6_2_22.AGAAGGAACA-TGGTAGATGC.HNGJJDSX3_AGAAGGAACA-TGGTAGATGC_L003_R2.fastq.gz fastq fastq 10483631624.0 34714012.0 GSM7963660 r1 0:151 1:151 A:2778161057;C:2394557806;G:2692864342;T:2618022344;N:26075 151 151     2778161057 2394557806 2692864342 2618022344 26075 SRX22832956 SRS19813396 SRA1765707 Pathology and Immunology, Washington University School of Medicine Pathology and Immunology, Washington University School of Medicine 2 0.91943 0.91641 0.08589 0.08581 0.78748 0.79774 0.5086 0.50263 151 151 B B biological fallback assumption illumina novaseq_era 3prime cdna_unspecified smarter bulk unknown unknown   United States 2023-12-08 Larval Larval Eye Sensory System
29120 29120 SRR27151355 SRX22832955 SRS19813395 SRP476687 PRJNA1050291 Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf td] GSE249755 Transcriptome Analysis The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 / retina red cones are transformed to hybrid red/UV sensitive cones green cones are transfated to blue cones and the number of rods is greatly reduced. In the mouse Samd7 / retina dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf thrb:tdTomato+ cells the following crosses were performed: WT × WT;thrb:tdTomato+/ or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS as described in another section. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total with RNA integrity RIN scores ranging from 7.4 8.4. parent bioproject:PRJNA1050288 pubmed:39531499   thrb:tdTomato+ cells from 5 dpf larval eyes samd7 / rep2 GSM7963659   source name:eye|cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / |geo loc name:missing|collection date:missing thrb:tdTomato+ cells from 5 dpf larval eyes samd7 / rep2 Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression DESeq2 1.34.0 was used in R 4.1.3 using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 / adult retina Table S3 we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 / and WT replicates derived from 5dpf thrb:tdTomato+ cells. Genes are ranked by p adj. eye   To perform RNA seq on 5 dpf thrb:tdTomato+ cells the following crosses were performed: WT × WT;thrb:tdTomato+/ or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / GSM7963659 GSM7963659: thrb:tdTomato+ cells from 5 dpf larval eyes samd7 / rep2; Danio rerio; RNA Seq GSM7963659 r1 GSM7963659 1 To perform RNA seq on 5 dpf thrb:tdTomato+ cells the following crosses were performed: WT × WT;thrb:tdTomato+/ or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP476687   loader:fastq load.py samd7_1_rna_6_1_22.AGACAGACGT-TATCAGTGCA.HNGJJDSX3_AGACAGACGT-TATCAGTGCA_L003_R1.fastq.gz samd7_1_rna_6_1_22.AGACAGACGT-TATCAGTGCA.HNGJJDSX3_AGACAGACGT-TATCAGTGCA_L003_R2.fastq.gz fastq fastq 12234446424.0 40511412.0 GSM7963659 r1 0:151 1:151 A:3397427491;C:2650252489;G:2963872812;T:3222862933;N:30699 151 151     3397427491 2650252489 2963872812 3222862933 30699 SRX22832955 SRS19813395 SRA1765707 Pathology and Immunology, Washington University School of Medicine Pathology and Immunology, Washington University School of Medicine 2 0.89702 0.8967 0.14358 0.14658 0.77309 0.77962 0.5468 0.44645 151 151 B B biological fallback assumption illumina novaseq_era 3prime cdna_unspecified smarter bulk unknown unknown   United States 2023-12-08 Larval Larval Eye Sensory System
29121 29121 SRR27151356 SRX22832954 SRS19813394 SRP476687 PRJNA1050291 Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf td] GSE249755 Transcriptome Analysis The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 / retina red cones are transformed to hybrid red/UV sensitive cones green cones are transfated to blue cones and the number of rods is greatly reduced. In the mouse Samd7 / retina dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf thrb:tdTomato+ cells the following crosses were performed: WT × WT;thrb:tdTomato+/ or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS as described in another section. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total with RNA integrity RIN scores ranging from 7.4 8.4. parent bioproject:PRJNA1050288 pubmed:39531499   thrb:tdTomato+ cells from 5 dpf larval eyes samd7 / rep1 GSM7963658   source name:eye|cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / |geo loc name:missing|collection date:missing thrb:tdTomato+ cells from 5 dpf larval eyes samd7 / rep1 Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression DESeq2 1.34.0 was used in R 4.1.3 using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 / adult retina Table S3 we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 / and WT replicates derived from 5dpf thrb:tdTomato+ cells. Genes are ranked by p adj. eye   To perform RNA seq on 5 dpf thrb:tdTomato+ cells the following crosses were performed: WT × WT;thrb:tdTomato+/ or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:samd7 / GSM7963658 GSM7963658: thrb:tdTomato+ cells from 5 dpf larval eyes samd7 / rep1; Danio rerio; RNA Seq GSM7963658 r1 GSM7963658 1 To perform RNA seq on 5 dpf thrb:tdTomato+ cells the following crosses were performed: WT × WT;thrb:tdTomato+/ or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP476687   loader:fastq load.py samd7_rna_5_10_22.CAATTCACGC-ACATCCTGCG.HNGJJDSX3_CAATTCACGC-ACATCCTGCG_L003_R1.fastq.gz samd7_rna_5_10_22.CAATTCACGC-ACATCCTGCG.HNGJJDSX3_CAATTCACGC-ACATCCTGCG_L003_R2.fastq.gz fastq fastq 11352128660.0 37589830.0 GSM7963658 r1 0:151 1:151 A:3035915425;C:2447970512;G:2970538003;T:2897676299;N:28421 151 151     3035915425 2447970512 2970538003 2897676299 28421 SRX22832954 SRS19813394 SRA1765707 Pathology and Immunology, Washington University School of Medicine Pathology and Immunology, Washington University School of Medicine 2 0.90706 0.91061 0.09833 0.102 0.78186 0.78948 0.54785 0.47215 151 151 B B biological fallback assumption illumina novaseq_era 3prime cdna_unspecified smarter bulk unknown unknown   United States 2023-12-08 Larval Larval Eye Sensory System
29122 29122 SRR27151357 SRX22832953 SRS19813393 SRP476687 PRJNA1050291 Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf td] GSE249755 Transcriptome Analysis The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 / retina red cones are transformed to hybrid red/UV sensitive cones green cones are transfated to blue cones and the number of rods is greatly reduced. In the mouse Samd7 / retina dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf thrb:tdTomato+ cells the following crosses were performed: WT × WT;thrb:tdTomato+/ or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS as described in another section. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total with RNA integrity RIN scores ranging from 7.4 8.4. parent bioproject:PRJNA1050288 pubmed:39531499   thrb:tdTomato+ cells from 5 dpf larval eyes WT rep3 GSM7963657   source name:eye|cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:WT|geo loc name:missing|collection date:missing thrb:tdTomato+ cells from 5 dpf larval eyes WT rep3 Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression DESeq2 1.34.0 was used in R 4.1.3 using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 / adult retina Table S3 we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 / and WT replicates derived from 5dpf thrb:tdTomato+ cells. Genes are ranked by p adj. eye   To perform RNA seq on 5 dpf thrb:tdTomato+ cells the following crosses were performed: WT × WT;thrb:tdTomato+/ or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:WT GSM7963657 GSM7963657: thrb:tdTomato+ cells from 5 dpf larval eyes WT rep3; Danio rerio; RNA Seq GSM7963657 r1 GSM7963657 1 To perform RNA seq on 5 dpf thrb:tdTomato+ cells the following crosses were performed: WT × WT;thrb:tdTomato+/ or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP476687   loader:fastq load.py wt_3_a_rna_5_12_22.CACCATTGTA-TACCAATTGC.HNGJJDSX3_CACCATTGTA-TACCAATTGC_L003_R1.fastq.gz wt_3_a_rna_5_12_22.CACCATTGTA-TACCAATTGC.HNGJJDSX3_CACCATTGTA-TACCAATTGC_L003_R2.fastq.gz fastq fastq 9723911364.0 32198382.0 GSM7963657 r1 0:151 1:151 A:2610813507;C:2104688166;G:2520224929;T:2488160661;N:24101 151 151     2610813507 2104688166 2520224929 2488160661 24101 SRX22832953 SRS19813393 SRA1765707 Pathology and Immunology, Washington University School of Medicine Pathology and Immunology, Washington University School of Medicine 2 0.62594 0.60745 0.07012 0.07039 0.81728 0.82578 0.50983 0.57347 151 151 B B biological fallback assumption illumina novaseq_era 3prime cdna_unspecified smarter bulk unknown unknown   United States 2023-12-08 Larval Larval Eye Sensory System
29123 29123 SRR27151358 SRX22832952 SRS19813392 SRP476687 PRJNA1050291 Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf td] GSE249755 Transcriptome Analysis The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 / retina red cones are transformed to hybrid red/UV sensitive cones green cones are transfated to blue cones and the number of rods is greatly reduced. In the mouse Samd7 / retina dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf thrb:tdTomato+ cells the following crosses were performed: WT × WT;thrb:tdTomato+/ or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS as described in another section. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total with RNA integrity RIN scores ranging from 7.4 8.4. parent bioproject:PRJNA1050288 pubmed:39531499   thrb:tdTomato+ cells from 5 dpf larval eyes WT rep2 GSM7963656   source name:eye|cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:WT|geo loc name:missing|collection date:missing thrb:tdTomato+ cells from 5 dpf larval eyes WT rep2 Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression DESeq2 1.34.0 was used in R 4.1.3 using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 / adult retina Table S3 we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 / and WT replicates derived from 5dpf thrb:tdTomato+ cells. Genes are ranked by p adj. eye   To perform RNA seq on 5 dpf thrb:tdTomato+ cells the following crosses were performed: WT × WT;thrb:tdTomato+/ or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:WT GSM7963656 GSM7963656: thrb:tdTomato+ cells from 5 dpf larval eyes WT rep2; Danio rerio; RNA Seq GSM7963656 r1 GSM7963656 1 To perform RNA seq on 5 dpf thrb:tdTomato+ cells the following crosses were performed: WT × WT;thrb:tdTomato+/ or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP476687   loader:fastq load.py wt_2_b_rna_5_12_22.AAGACTCTCG-TAAGTCTGGC.HNGJJDSX3_AAGACTCTCG-TAAGTCTGGC_L003_R1.fastq.gz wt_2_b_rna_5_12_22.AAGACTCTCG-TAAGTCTGGC.HNGJJDSX3_AAGACTCTCG-TAAGTCTGGC_L003_R2.fastq.gz fastq fastq 10722667946.0 35505523.0 GSM7963656 r1 0:151 1:151 A:2872256957;C:2358147851;G:2771142656;T:2721093653;N:26829 151 151     2872256957 2358147851 2771142656 2721093653 26829 SRX22832952 SRS19813392 SRA1765707 Pathology and Immunology, Washington University School of Medicine Pathology and Immunology, Washington University School of Medicine 2 0.90028 0.8986 0.10012 0.10225 0.7973 0.80389 0.56209 0.56007 151 151 B B biological fallback assumption illumina novaseq_era 3prime cdna_unspecified smarter bulk unknown unknown   United States 2023-12-08 Larval Larval Eye Sensory System
29124 29124 SRR27151359 SRX22832951 SRS19813391 SRP476687 PRJNA1050291 Samd7 preserves cell identity and enforces the 'one neuron one receptor' rule in vertebrate photoreceptors [5dpf td] GSE249755 Transcriptome Analysis The exclusive expression of single sensory receptors in individual neurons the 'one neuron one receptor' rule is essential for vision and other sensory systems. Here we show that the transcriptional corepressor Samd7 enforces this rule in vertebrate red cones and acts in other photoreceptor types to maintain cell identity. In the zebrafish samd7 / retina red cones are transformed to hybrid red/UV sensitive cones green cones are transfated to blue cones and the number of rods is greatly reduced. In the mouse Samd7 / retina dorsal M cones are transformed to hybrid M/S cones—analogous to the transformation of red to red/UV cones that occurs in zebrafish—and rods aberrantly express cone genes including S opsin. Altogether Samd7 acts to repress short wavelength cone gene expression in long wavelength sensitive cones thereby sustaining the mutually exclusive patterns of opsin expression and cone identity required for color vision. Overall design: To perform RNA seq on 5 dpf thrb:tdTomato+ cells the following crosses were performed: WT × WT;thrb:tdTomato+/ or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS as described in another section. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total with RNA integrity RIN scores ranging from 7.4 8.4. parent bioproject:PRJNA1050288 pubmed:39531499   thrb:tdTomato+ cells from 5 dpf larval eyes WT rep1 GSM7963655   source name:eye|cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:WT|geo loc name:missing|collection date:missing thrb:tdTomato+ cells from 5 dpf larval eyes WT rep1 Sequencing adapters were trimmed using trimgalore 0.6.1 https://github.com/FelixKrueger/TrimGalore61. RNA seq reads were then aligned to danRer10 using STAR 2.7.2b with an index prepared for 150 bp reads62. Next Htseq 0.9.1 was used to generate normalized read counts63. To calculate differential gene expression DESeq2 1.34.0 was used in R 4.1.3 using a log2 fold change threshold of 0 and an FDR p adj of 0.164. Volcano plots were then labeled to identify genes with p adj < 0.05. To identify genes as photoreceptor subtype specific manual curation was performed using publicly available RNA seq data from adult zebrafish photoreceptor subtypes4 5. To identify rod specific gene dysregulated in the samd7 / adult retina Table S3 we manually cross referenced the top 40 most enriched rod specific genes and mafba from Ogawa et al5. Assembly: danRer10 Supplementary files format and content: csv file of DESeq2 normalized counts and averages from three samd7 / and WT replicates derived from 5dpf thrb:tdTomato+ cells. Genes are ranked by p adj. eye   To perform RNA seq on 5 dpf thrb:tdTomato+ cells the following crosses were performed: WT × WT;thrb:tdTomato+/ or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer’s protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the 3’ ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   cell type:thrb:tdTomato+ cells|tissue:eye|developmental stage:5 dpf larvae|genotype:WT GSM7963655 GSM7963655: thrb:tdTomato+ cells from 5 dpf larval eyes WT rep1; Danio rerio; RNA Seq GSM7963655 r1 GSM7963655 1 To perform RNA seq on 5 dpf thrb:tdTomato+ cells the following crosses were performed: WT × WT;thrb:tdTomato+/ or samd7stl888/stl888 × samd7stl888/stl888;thrb:tdTomato+/ . A subset of larvae were genotyped from each resultant clutch. TdTomato+ larvae were then separated for subsequent dissociation. 40 50 eyes from 20 25 larvae were dissected from each clutch as an individual replicate. The eyes were then dissociated into single cells and subjected to FACS. 10 000 25 000 cells were collected and sorted directly to buffer RLT from the Rneasy minElute Cleanup Kit Qiagen; RNA was then extracted using the Rneasy Micro Kit Qiagen and on column DNAse treatment performed using the Rnase Free Dnase Set Qiagen. RNA concentrations ranged from 40 190 pg/µl 0.3 1.8 ng total with RNA integrity RIN scores ranging from 7.4 8.4. Library preparation was performed with 10 ng of total RNA for 5 dpf whole eye and adult retina samples and with 300 pg of total RNA for 5 dpf thrb:tdTomato+ cells. Double stranded cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing Takara Clontech per manufacturer's protocol using 12 amplification cycles for 5 dpf whole eye and adult retina samples and 14 amplification cycles for 5 dpf thrb:tdTomato+ cell samples. cDNA was fragmented using a Covaris E220 sonicator with peak incident power 18 duty factor 20% cycles per burst 50 for 120 seconds. cDNA was blunt ended using a combination of T4 DNA Polymerase Klenow Fragment DNA Polymerase and T4 PolyNucleotide Kinase; an A base was added to the three prime ends using Klenow three prime five prime exo and Illumina sequencing adapters were ligated to the ends using T4 DNA ligase Qiagen Enzymatics. Ligated fragments were then amplified for 12 cycles for 5 dpf whole eye samples and 15 cycles for adult retina and 5 dpf thrb:tdTomato+ cell samples using primers incorporating unique dual index tags using 2X VeraSeq PCR mix. DNA was sequenced on an Illumina NovaSeq 6000 using 150 bp paired end reads.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP476687   loader:fastq load.py wt_rna_5_10_22.CCACCTTCAA-ACCATCGAGG.HNGJJDSX3_CCACCTTCAA-ACCATCGAGG_L003_R1.fastq.gz wt_rna_5_10_22.CCACCTTCAA-ACCATCGAGG.HNGJJDSX3_CCACCTTCAA-ACCATCGAGG_L003_R2.fastq.gz fastq fastq 9875387618.0 32699959.0 GSM7963655 r1 0:151 1:151 A:2640364023;C:2083842037;G:2621465276;T:2529691405;N:24877 151 151     2640364023 2083842037 2621465276 2529691405 24877 SRX22832951 SRS19813391 SRA1765707 Pathology and Immunology, Washington University School of Medicine Pathology and Immunology, Washington University School of Medicine 2 0.90131 0.90429 0.10837 0.1106 0.79523 0.80182 0.50934 0.58039 151 151 B B biological fallback assumption illumina novaseq_era 3prime cdna_unspecified smarter bulk unknown unknown   United States 2023-12-08 Larval Larval Eye Sensory System
29174 29174 SRR27237224 SRX22915658 SRS19883761 SRP478464 PRJNA1053781 Single cell gene expression profie of developing photoreceptor cells in larval zebrafish GSE250379 Other Molecular underpinnings of vertebrate retinal differentiation and maturation are poorly understood particularly for non mammalian species. We generated single cell transcriptome data from the larval zebrafish retina and characterized gene expression diversity among photoreceptor subtypes and their progenitors. Overall design: GFP positive differentiating photoreceptor cells and bipolar cells were collected from 4 dpf larval transgenic zebrafish Tgcrx:EGFPstl887 using fluorescence activated cell sorting. parent bioproject:PRJNA1050288 pubmed:39531499   retina scRNA seq GSM7978132   source name:retina|tissue:retina|genotype:Tgcrx:EGFPstl887|developmental stage:4 dpf|geo loc name:missing|collection date:missing retina scRNA seq Read alignment and initial quality control were performed using Cell Ranger software version 7.0.0 10X Genomics. Assembly: GRCz11 Supplementary files format and content: Tab separated value file and matrix file retina   Fifty heads were dissected from 4 dpf heterozygous Tgcrx:GFPstl887Tg larvae. Following dissection eyes were stored in ice cold Hanks’ Balanced Salt Solution HBSS until all eyes were harvested. Once the eyes were collected HBSS was removed and the eyes were incubated in 400 µl of calcium/magnesium free HBSS containing 0.4 mg papain Worthington Biochem for 15 min at 37°C. 800 µl of 10% fetal bovine serum FBS in Dulbecco's Modified Eagle Medium DMEM containing 5mM MgCl2 and 120 units DNaseI Roche were added to the mixture and incubated for 5 min at 37°C. Cells were then resuspended in 300 µl of sorting buffer 2.5 mM EDTA 25 mM HEPES 1% bovine serum albumin BSA in calcium/magnesium free HBSS. Cells were sorted on an Aria II FACS machine BD biosciences with gating based on forward scatter side scatter and GFP fluorescence and collected in 700 μl of D PBS without xxx+ and Mg2+ supplemented with 0.4 % BSA D PBS CMF in 1.5 ml microcentrifuge tubes. The collected cells were then centrifuged at 300×g for 5 min washed with D PBS CMF centrifuged and supernatant reduced to 80 µl. Cell density was quantified on a hemocytometer and 5000 cells were used for single cell library preparation. A library for single cell RNA seq was constructed with the Chromium v3 platform 10X Genomics Pleasanton CA according to the manufacturer protocol.   tissue:retina|genotype:Tgcrx:EGFPstl887|developmental stage:4 dpf GSM7978132 GSM7978132: retina scRNA seq; Danio rerio; RNA Seq GSM7978132 r1 GSM7978132 1 Fifty heads were dissected from 4 dpf heterozygous Tgcrx:GFPstl887Tg larvae. Following dissection eyes were stored in ice cold Hanks' Balanced Salt Solution HBSS until all eyes were harvested. Once the eyes were collected HBSS was removed and the eyes were incubated in 400 µl of calcium/magnesium free HBSS containing 0.4 mg papain Worthington Biochem for 15 min at 37°C. 800 µl of 10% fetal bovine serum FBS in Dulbecco's Modified Eagle Medium DMEM containing 5mM MgCl2 and 120 units DNaseI Roche were added to the mixture and incubated for 5 min at 37°C. Cells were then resuspended in 300 µl of sorting buffer 2.5 mM EDTA 25 mM HEPES 1% bovine serum albumin BSA in calcium/magnesium free HBSS. Cells were sorted on an Aria II FACS machine BD biosciences with gating based on forward scatter side scatter and GFP fluorescence and collected in 700 μl of D PBS without xxx+ and Mg2+ supplemented with 0.4 % BSA D PBS CMF in 1.5 ml microcentrifuge tubes. The collected cells were then centrifuged at 300×g for 5 min washed with D PBS CMF centrifuged and supernatant reduced to 80 µl. Cell density was quantified on a hemocytometer and 5000 cells were used for single cell library preparation. A library for single cell RNA seq was constructed with the Chromium v3 platform 10X Genomics Pleasanton CA according to the manufacturer protocol.   RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA PAIRED ILLUMINA Illumina NovaSeq 6000   SRP478464   loader:fastq load.py crx.crx_S1_L003_R1_001.fastq.gz crx.crx_S1_L003_R2_001.fastq.gz fastq fastq 45170138494.0 253764823.0 GSM7978132 r1 0:28 1:150 A:13826023801;C:9023674621;G:9590649474;T:12728940802;N:849796 28 150     13826023801 9023674621 9590649474 12728940802 849796 SRX22915658 SRS19883761 SRA1770358 Pathology and Immunology, Washington University School of Medicine Pathology and Immunology, Washington University School of Medicine 2 0.0046 0.87155 0.00206 0.2263 0.99168 0.77784 0.30223 0.50112 28 150 T B sc-like readlen illumina novaseq_era unknown cdna_unspecified unknown sc single_cell_droplet 10x   United States 2023-12-17 Larval Larval Eye Sensory System
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
31849 31849 SRR28735527 SRX24301508 SRS21065427 SRP502615 PRJNA1101945 Loss of Stim2 in zebrafish induces retinal gene expression cellular changes resembling glaucoma characteristics [RNA seq] GSE264309 Transcriptome Analysis Calcium is involved in vision processes in retina and is implicated in various pathologies including glaucoma. Rods are protected against prolonged lowering of intracellular calcium ion concentrations by Store Operated Calcium Entry SOCE. We showed zebrafish lacking SOCE calcium sensor Stim2 had problem with vision as indicated by behavior tests staining of retina and downregulation of genes related to light perception. In this work we aimed to understand the mechanism responsible for the vision problems in stim2 zebrafish knockout. scRNA sequencing of neuronal origin cells from brains of 5 dpf larvae identified 27 clusters. Differently expressed genes were detected in ten clusters including amacrine and GABAergic retinal interneurons and GABAergic optic tectum cells. In five clusters the proportion of cells in stim2 KO fish versus control was significantly decreased including GABAergic diencephalon and optic tectum and was increased in amacrine and GABAergic retinal interneurons. Transmission Electron Microscopy in stim2 KO fish revealed decrease in width of inner plexiform layer IPL ganglion cells and their dendrites numbers what is characteristic for glaucoma. Analysis of cell density in the inner nucleus layer which includes amacrine cells among others showed a significant decrease in the number of GABAergic neurons. The area of cristae in photoreceptor mitochondria was statistically lower in stim2 KO than in control retinas. Overall design: AB and double knockout for stim2 stim2a;stim2b / zebrafish 5dpf larvae were used to isolate the total RNA from their eyes. The experimental setup involved 3 replicates per each condition stim2 KO vs control consisting of 35 eyes per repetition. parent bioproject:PRJNA1101942 pubmed:39424970   stim2KO repllicate 3 RNASeq GSM8215878   source name:eye|tissue:eye|genotype:stim2a;stim2b / |age:5 dpf|geo loc name:missing|collection date:missing stim2KO repllicate 3 RNASeq Raw RNA seq data were converted to FASTQ format with bcl2fastq2 v2.17 Illumina and FastQC v0.11.4 was used to assess the quality of each sequencing run. We then utilized the SQuIRE pipeline45 for subsequent steps. STAR version 2.5.3a StringTie version 1.3.3b and DESeq2 version 1.16.149 were used within the SQuIRE pipeline for read alignment transcript assembly and quantification for each replicate and differential gene expression analysis respectively. Assembly: Danio rerio GRCz11 Supplementary files format and content: Tab separated files with raw reads for each sample eye   Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260 280 and 230 nm and Bioanalyzer Nano RNA chip Agilent USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research Irvine CA USA according to the manufacturer’s recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.   tissue:eye|genotype:stim2a;stim2b / |age:5 dpf GSM8215878 GSM8215878: stim2KO repllicate 3 RNASeq; Danio rerio; RNA Seq GSM8215878 r1 GSM8215878 1 Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260 280 and 230 nm and Bioanalyzer Nano RNA chip Agilent USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research Irvine CA USA according to the manufacturer's recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 500   SRP502615   loader:fastq load.py mut_rep_3_R1_001.fastq.gz mut_rep_3_R2_001.fastq.gz fastq fastq 5198552618.0 25735409.0 GSM8215878 r1 0:101 1:101 A:1302884238;C:1298420335;G:1334893927;T:1262296331;N:57787 101 101     1302884238 1298420335 1334893927 1262296331 57787 SRX24301508 SRS21065427 SRA1848589 Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS 2 0.95719 0.95856 0.05756 0.05712 0.70534 0.70851 0.42046 0.41786 101 101 B B biological fallback assumption illumina nextseq unknown cdna_unspecified trueseq sc_generic single_cell_generic generic-scrnaseq-only   Poland 2024-04-18 Larval Larval Eye Sensory System
31850 31850 SRR28735528 SRX24301507 SRS21065426 SRP502615 PRJNA1101945 Loss of Stim2 in zebrafish induces retinal gene expression cellular changes resembling glaucoma characteristics [RNA seq] GSE264309 Transcriptome Analysis Calcium is involved in vision processes in retina and is implicated in various pathologies including glaucoma. Rods are protected against prolonged lowering of intracellular calcium ion concentrations by Store Operated Calcium Entry SOCE. We showed zebrafish lacking SOCE calcium sensor Stim2 had problem with vision as indicated by behavior tests staining of retina and downregulation of genes related to light perception. In this work we aimed to understand the mechanism responsible for the vision problems in stim2 zebrafish knockout. scRNA sequencing of neuronal origin cells from brains of 5 dpf larvae identified 27 clusters. Differently expressed genes were detected in ten clusters including amacrine and GABAergic retinal interneurons and GABAergic optic tectum cells. In five clusters the proportion of cells in stim2 KO fish versus control was significantly decreased including GABAergic diencephalon and optic tectum and was increased in amacrine and GABAergic retinal interneurons. Transmission Electron Microscopy in stim2 KO fish revealed decrease in width of inner plexiform layer IPL ganglion cells and their dendrites numbers what is characteristic for glaucoma. Analysis of cell density in the inner nucleus layer which includes amacrine cells among others showed a significant decrease in the number of GABAergic neurons. The area of cristae in photoreceptor mitochondria was statistically lower in stim2 KO than in control retinas. Overall design: AB and double knockout for stim2 stim2a;stim2b / zebrafish 5dpf larvae were used to isolate the total RNA from their eyes. The experimental setup involved 3 replicates per each condition stim2 KO vs control consisting of 35 eyes per repetition. parent bioproject:PRJNA1101942 pubmed:39424970   stim2KO repllicate 2 RNASeq GSM8215877   source name:eye|tissue:eye|genotype:stim2a;stim2b / |age:5 dpf|geo loc name:missing|collection date:missing stim2KO repllicate 2 RNASeq Raw RNA seq data were converted to FASTQ format with bcl2fastq2 v2.17 Illumina and FastQC v0.11.4 was used to assess the quality of each sequencing run. We then utilized the SQuIRE pipeline45 for subsequent steps. STAR version 2.5.3a StringTie version 1.3.3b and DESeq2 version 1.16.149 were used within the SQuIRE pipeline for read alignment transcript assembly and quantification for each replicate and differential gene expression analysis respectively. Assembly: Danio rerio GRCz11 Supplementary files format and content: Tab separated files with raw reads for each sample eye   Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260 280 and 230 nm and Bioanalyzer Nano RNA chip Agilent USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research Irvine CA USA according to the manufacturer’s recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.   tissue:eye|genotype:stim2a;stim2b / |age:5 dpf GSM8215877 GSM8215877: stim2KO repllicate 2 RNASeq; Danio rerio; RNA Seq GSM8215877 r1 GSM8215877 1 Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260 280 and 230 nm and Bioanalyzer Nano RNA chip Agilent USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research Irvine CA USA according to the manufacturer's recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 500   SRP502615   loader:fastq load.py mut_rep_2_R1_001.fastq.gz mut_rep_2_R2_001.fastq.gz fastq fastq 5641008570.0 27925785.0 GSM8215877 r1 0:101 1:101 A:1421655053;C:1399106135;G:1439454285;T:1380731092;N:62005 101 101     1421655053 1399106135 1439454285 1380731092 62005 SRX24301507 SRS21065426 SRA1848589 Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS 2 0.95794 0.9601 0.05457 0.05365 0.70942 0.71151 0.42408 0.42666 101 101 B B biological fallback assumption illumina nextseq unknown cdna_unspecified trueseq sc_generic single_cell_generic generic-scrnaseq-only   Poland 2024-04-18 Larval Larval Eye Sensory System
31851 31851 SRR28735529 SRX24301506 SRS21065425 SRP502615 PRJNA1101945 Loss of Stim2 in zebrafish induces retinal gene expression cellular changes resembling glaucoma characteristics [RNA seq] GSE264309 Transcriptome Analysis Calcium is involved in vision processes in retina and is implicated in various pathologies including glaucoma. Rods are protected against prolonged lowering of intracellular calcium ion concentrations by Store Operated Calcium Entry SOCE. We showed zebrafish lacking SOCE calcium sensor Stim2 had problem with vision as indicated by behavior tests staining of retina and downregulation of genes related to light perception. In this work we aimed to understand the mechanism responsible for the vision problems in stim2 zebrafish knockout. scRNA sequencing of neuronal origin cells from brains of 5 dpf larvae identified 27 clusters. Differently expressed genes were detected in ten clusters including amacrine and GABAergic retinal interneurons and GABAergic optic tectum cells. In five clusters the proportion of cells in stim2 KO fish versus control was significantly decreased including GABAergic diencephalon and optic tectum and was increased in amacrine and GABAergic retinal interneurons. Transmission Electron Microscopy in stim2 KO fish revealed decrease in width of inner plexiform layer IPL ganglion cells and their dendrites numbers what is characteristic for glaucoma. Analysis of cell density in the inner nucleus layer which includes amacrine cells among others showed a significant decrease in the number of GABAergic neurons. The area of cristae in photoreceptor mitochondria was statistically lower in stim2 KO than in control retinas. Overall design: AB and double knockout for stim2 stim2a;stim2b / zebrafish 5dpf larvae were used to isolate the total RNA from their eyes. The experimental setup involved 3 replicates per each condition stim2 KO vs control consisting of 35 eyes per repetition. parent bioproject:PRJNA1101942 pubmed:39424970   stim2KO repllicate 1 RNASeq GSM8215876   source name:eye|tissue:eye|genotype:stim2a;stim2b / |age:5 dpf|geo loc name:missing|collection date:missing stim2KO repllicate 1 RNASeq Raw RNA seq data were converted to FASTQ format with bcl2fastq2 v2.17 Illumina and FastQC v0.11.4 was used to assess the quality of each sequencing run. We then utilized the SQuIRE pipeline45 for subsequent steps. STAR version 2.5.3a StringTie version 1.3.3b and DESeq2 version 1.16.149 were used within the SQuIRE pipeline for read alignment transcript assembly and quantification for each replicate and differential gene expression analysis respectively. Assembly: Danio rerio GRCz11 Supplementary files format and content: Tab separated files with raw reads for each sample eye   Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260 280 and 230 nm and Bioanalyzer Nano RNA chip Agilent USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research Irvine CA USA according to the manufacturer’s recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.   tissue:eye|genotype:stim2a;stim2b / |age:5 dpf GSM8215876 GSM8215876: stim2KO repllicate 1 RNASeq; Danio rerio; RNA Seq GSM8215876 r1 GSM8215876 1 Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260 280 and 230 nm and Bioanalyzer Nano RNA chip Agilent USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research Irvine CA USA according to the manufacturer's recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 500   SRP502615   loader:fastq load.py mut_rep_1_R1_001.fastq.gz mut_rep_1_R2_001.fastq.gz fastq fastq 5147003026.0 25480213.0 GSM8215876 r1 0:101 1:101 A:1302966446;C:1274828722;G:1325578656;T:1243571921;N:57281 101 101     1302966446 1274828722 1325578656 1243571921 57281 SRX24301506 SRS21065425 SRA1848589 Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS 2 0.95963 0.96036 0.05042 0.05053 0.71275 0.71725 0.41458 0.41703 101 101 B B biological fallback assumption illumina nextseq unknown cdna_unspecified trueseq sc_generic single_cell_generic generic-scrnaseq-only   Poland 2024-04-18 Larval Larval Eye Sensory System
31852 31852 SRR28735530 SRX24301505 SRS21065424 SRP502615 PRJNA1101945 Loss of Stim2 in zebrafish induces retinal gene expression cellular changes resembling glaucoma characteristics [RNA seq] GSE264309 Transcriptome Analysis Calcium is involved in vision processes in retina and is implicated in various pathologies including glaucoma. Rods are protected against prolonged lowering of intracellular calcium ion concentrations by Store Operated Calcium Entry SOCE. We showed zebrafish lacking SOCE calcium sensor Stim2 had problem with vision as indicated by behavior tests staining of retina and downregulation of genes related to light perception. In this work we aimed to understand the mechanism responsible for the vision problems in stim2 zebrafish knockout. scRNA sequencing of neuronal origin cells from brains of 5 dpf larvae identified 27 clusters. Differently expressed genes were detected in ten clusters including amacrine and GABAergic retinal interneurons and GABAergic optic tectum cells. In five clusters the proportion of cells in stim2 KO fish versus control was significantly decreased including GABAergic diencephalon and optic tectum and was increased in amacrine and GABAergic retinal interneurons. Transmission Electron Microscopy in stim2 KO fish revealed decrease in width of inner plexiform layer IPL ganglion cells and their dendrites numbers what is characteristic for glaucoma. Analysis of cell density in the inner nucleus layer which includes amacrine cells among others showed a significant decrease in the number of GABAergic neurons. The area of cristae in photoreceptor mitochondria was statistically lower in stim2 KO than in control retinas. Overall design: AB and double knockout for stim2 stim2a;stim2b / zebrafish 5dpf larvae were used to isolate the total RNA from their eyes. The experimental setup involved 3 replicates per each condition stim2 KO vs control consisting of 35 eyes per repetition. parent bioproject:PRJNA1101942 pubmed:39424970   Tg repllicate 3 RNASeq GSM8215875   source name:eye|tissue:eye|genotype:AB|age:5 dpf|geo loc name:missing|collection date:missing Tg repllicate 3 RNASeq Raw RNA seq data were converted to FASTQ format with bcl2fastq2 v2.17 Illumina and FastQC v0.11.4 was used to assess the quality of each sequencing run. We then utilized the SQuIRE pipeline45 for subsequent steps. STAR version 2.5.3a StringTie version 1.3.3b and DESeq2 version 1.16.149 were used within the SQuIRE pipeline for read alignment transcript assembly and quantification for each replicate and differential gene expression analysis respectively. Assembly: Danio rerio GRCz11 Supplementary files format and content: Tab separated files with raw reads for each sample eye   Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260 280 and 230 nm and Bioanalyzer Nano RNA chip Agilent USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research Irvine CA USA according to the manufacturer’s recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.   tissue:eye|genotype:AB|age:5 dpf GSM8215875 GSM8215875: Tg repllicate 3 RNASeq; Danio rerio; RNA Seq GSM8215875 r1 GSM8215875 1 Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260 280 and 230 nm and Bioanalyzer Nano RNA chip Agilent USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research Irvine CA USA according to the manufacturer's recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 500   SRP502615   loader:fastq load.py wt_rep_3_R1_001.fastq.gz wt_rep_3_R2_001.fastq.gz fastq fastq 4648345422.0 23011611.0 GSM8215875 r1 0:101 1:101 A:1186641846;C:1144216110;G:1192801277;T:1124633797;N:52392 101 101     1186641846 1144216110 1192801277 1124633797 52392 SRX24301505 SRS21065424 SRA1848589 Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS 2 0.9575 0.95756 0.05359 0.05311 0.69875 0.70315 0.42041 0.41054 101 101 B B biological fallback assumption illumina nextseq unknown cdna_unspecified trueseq sc_generic single_cell_generic generic-scrnaseq-only   Poland 2024-04-18 Larval Larval Eye Sensory System
31853 31853 SRR28735531 SRX24301504 SRS21065423 SRP502615 PRJNA1101945 Loss of Stim2 in zebrafish induces retinal gene expression cellular changes resembling glaucoma characteristics [RNA seq] GSE264309 Transcriptome Analysis Calcium is involved in vision processes in retina and is implicated in various pathologies including glaucoma. Rods are protected against prolonged lowering of intracellular calcium ion concentrations by Store Operated Calcium Entry SOCE. We showed zebrafish lacking SOCE calcium sensor Stim2 had problem with vision as indicated by behavior tests staining of retina and downregulation of genes related to light perception. In this work we aimed to understand the mechanism responsible for the vision problems in stim2 zebrafish knockout. scRNA sequencing of neuronal origin cells from brains of 5 dpf larvae identified 27 clusters. Differently expressed genes were detected in ten clusters including amacrine and GABAergic retinal interneurons and GABAergic optic tectum cells. In five clusters the proportion of cells in stim2 KO fish versus control was significantly decreased including GABAergic diencephalon and optic tectum and was increased in amacrine and GABAergic retinal interneurons. Transmission Electron Microscopy in stim2 KO fish revealed decrease in width of inner plexiform layer IPL ganglion cells and their dendrites numbers what is characteristic for glaucoma. Analysis of cell density in the inner nucleus layer which includes amacrine cells among others showed a significant decrease in the number of GABAergic neurons. The area of cristae in photoreceptor mitochondria was statistically lower in stim2 KO than in control retinas. Overall design: AB and double knockout for stim2 stim2a;stim2b / zebrafish 5dpf larvae were used to isolate the total RNA from their eyes. The experimental setup involved 3 replicates per each condition stim2 KO vs control consisting of 35 eyes per repetition. parent bioproject:PRJNA1101942 pubmed:39424970   Tg repllicate 2 RNASeq GSM8215874   source name:eye|tissue:eye|genotype:AB|age:5 dpf|geo loc name:missing|collection date:missing Tg repllicate 2 RNASeq Raw RNA seq data were converted to FASTQ format with bcl2fastq2 v2.17 Illumina and FastQC v0.11.4 was used to assess the quality of each sequencing run. We then utilized the SQuIRE pipeline45 for subsequent steps. STAR version 2.5.3a StringTie version 1.3.3b and DESeq2 version 1.16.149 were used within the SQuIRE pipeline for read alignment transcript assembly and quantification for each replicate and differential gene expression analysis respectively. Assembly: Danio rerio GRCz11 Supplementary files format and content: Tab separated files with raw reads for each sample eye   Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260 280 and 230 nm and Bioanalyzer Nano RNA chip Agilent USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research Irvine CA USA according to the manufacturer’s recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.   tissue:eye|genotype:AB|age:5 dpf GSM8215874 GSM8215874: Tg repllicate 2 RNASeq; Danio rerio; RNA Seq GSM8215874 r1 GSM8215874 1 Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260 280 and 230 nm and Bioanalyzer Nano RNA chip Agilent USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research Irvine CA USA according to the manufacturer's recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 500   SRP502615   loader:fastq load.py wt_rep_2_R1_001.fastq.gz wt_rep_2_R2_001.fastq.gz fastq fastq 4769618546.0 23611973.0 GSM8215874 r1 0:101 1:101 A:1218890346;C:1171831021;G:1228410692;T:1150434539;N:51948 101 101     1218890346 1171831021 1228410692 1150434539 51948 SRX24301504 SRS21065423 SRA1848589 Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS 2 0.95682 0.95517 0.05192 0.05059 0.70849 0.71543 0.41181 0.41549 101 101 B B biological fallback assumption illumina nextseq unknown cdna_unspecified trueseq sc_generic single_cell_generic generic-scrnaseq-only   Poland 2024-04-18 Larval Larval Eye Sensory System
31854 31854 SRR28735532 SRX24301503 SRS21065422 SRP502615 PRJNA1101945 Loss of Stim2 in zebrafish induces retinal gene expression cellular changes resembling glaucoma characteristics [RNA seq] GSE264309 Transcriptome Analysis Calcium is involved in vision processes in retina and is implicated in various pathologies including glaucoma. Rods are protected against prolonged lowering of intracellular calcium ion concentrations by Store Operated Calcium Entry SOCE. We showed zebrafish lacking SOCE calcium sensor Stim2 had problem with vision as indicated by behavior tests staining of retina and downregulation of genes related to light perception. In this work we aimed to understand the mechanism responsible for the vision problems in stim2 zebrafish knockout. scRNA sequencing of neuronal origin cells from brains of 5 dpf larvae identified 27 clusters. Differently expressed genes were detected in ten clusters including amacrine and GABAergic retinal interneurons and GABAergic optic tectum cells. In five clusters the proportion of cells in stim2 KO fish versus control was significantly decreased including GABAergic diencephalon and optic tectum and was increased in amacrine and GABAergic retinal interneurons. Transmission Electron Microscopy in stim2 KO fish revealed decrease in width of inner plexiform layer IPL ganglion cells and their dendrites numbers what is characteristic for glaucoma. Analysis of cell density in the inner nucleus layer which includes amacrine cells among others showed a significant decrease in the number of GABAergic neurons. The area of cristae in photoreceptor mitochondria was statistically lower in stim2 KO than in control retinas. Overall design: AB and double knockout for stim2 stim2a;stim2b / zebrafish 5dpf larvae were used to isolate the total RNA from their eyes. The experimental setup involved 3 replicates per each condition stim2 KO vs control consisting of 35 eyes per repetition. parent bioproject:PRJNA1101942 pubmed:39424970   Tg repllicate 1 RNASeq GSM8215873   source name:eye|tissue:eye|genotype:AB|age:5 dpf|geo loc name:missing|collection date:missing Tg repllicate 1 RNASeq Raw RNA seq data were converted to FASTQ format with bcl2fastq2 v2.17 Illumina and FastQC v0.11.4 was used to assess the quality of each sequencing run. We then utilized the SQuIRE pipeline45 for subsequent steps. STAR version 2.5.3a StringTie version 1.3.3b and DESeq2 version 1.16.149 were used within the SQuIRE pipeline for read alignment transcript assembly and quantification for each replicate and differential gene expression analysis respectively. Assembly: Danio rerio GRCz11 Supplementary files format and content: Tab separated files with raw reads for each sample eye   Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260 280 and 230 nm and Bioanalyzer Nano RNA chip Agilent USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research Irvine CA USA according to the manufacturer’s recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.   tissue:eye|genotype:AB|age:5 dpf GSM8215873 GSM8215873: Tg repllicate 1 RNASeq; Danio rerio; RNA Seq GSM8215873 r1 GSM8215873 1 Total RNA was isolated for RNA seq using TRIzol reagent. Thirty five larvae were pooled together to comprise one RNA sample. The quality of RNA was checked by measuring absorbance at 260 280 and 230 nm and Bioanalyzer Nano RNA chip Agilent USA analysis. Samples with 260/280 nm and 230/280 nm absorbance ratio >1.8 and RNA integrity number values >8 were used for further processing. Total RNA was first digested by DNase I and purified using the RNA Clean and Concentrator Kit ZYMO Research Irvine CA USA according to the manufacturer's recommendations. Library construction was conducted using stranded Illumina TruSeq protocol.   RNA-Seq TRANSCRIPTOMIC cDNA PAIRED ILLUMINA NextSeq 500   SRP502615   loader:fastq load.py wt_rep_1_R1_001.fastq.gz wt_rep_1_R2_001.fastq.gz fastq fastq 4771981138.0 23623669.0 GSM8215873 r1 0:101 1:101 A:1222918015;C:1171619846;G:1231393968;T:1145997219;N:52090 101 101     1222918015 1171619846 1231393968 1145997219 52090 SRX24301503 SRS21065422 SRA1848589 Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS 2 0.95739 0.95621 0.05362 0.0539 0.71301 0.71979 0.42337 0.40836 101 101 B B biological fallback assumption illumina nextseq unknown cdna_unspecified trueseq sc_generic single_cell_generic generic-scrnaseq-only   Poland 2024-04-18 Larval Larval Eye Sensory System
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

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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");;