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298 rows where devstage_curation = "Larval" and experiment.library_selection = "other"

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tissue_curation 7

technology 7

experiment.library_strategy 5

experiment.platform 3

experiment.library_source 2

experiment.library_layout 2

experiment.library_selection 1

  • other · 298 ✖

devstage_curation_coarse 1

devstage_curation 1

  • Larval · 298 ✖
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
75 75 DRR032749 DRX029555 DRS049954 DRP003810 PRJDB3785 EXPANDE project DRP003810 Other EXPression AloNg Development and Evolution EXPANDE project aims to identify gene expression profiles expanded during embryogenesis and evolution. In brief taking advantages of Illumina sequencing RNAseq profiles of early to late embryos of 8 chordate species were identified with biological replicates two or more biological replicates.     mRNA extracted from pooled embryos of 50 individuals Dr 72h 2 SAMD00028146   sample name:Dr 72h 2|strain:Riken WT Wild Type|tissue type:whole embryo|dev stage:72h Protruding mouth|genotype:wild type|phenotype:wild type|sex:male female and mixed                 Illumina HiSeq 2000 sequencing of SAMD00028146 DRX029555 Dr 72h 2 1 Total RNA QIAGEN RNeasy followed by TruSeq   RNA-Seq TRANSCRIPTOMIC other SINGLE ILLUMINA Illumina HiSeq 2000 <SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>100</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR> DRP003810 Illumina HiSeq 2000 sequencing of SAMD00028146       3429795800.0 34297958.0 DRR032749 0:100 1:0 A:928062015;C:792470305;G:786930881;T:922296289;N:36310 100 0     928062015 792470305 786930881 922296289 36310 DRX029555 DRS049954 DRA003460 UT-BS|Lab for embryology, Department of Biological Sciences, University of Tokyo UT-BS|Lab for embryology, Department of Biological Sciences, University of Tokyo 1 0.91821   0.09774   0.65437   0.46443   100   B   usable mapping rate illumina hiseq_era unknown other trueseq bulk unknown unknown   Japan 2017-09-20 Larval Larval Whole Organism All anatomical structures
76 76 DRR032748 DRX029554 DRS049953 DRP003810 PRJDB3785 EXPANDE project DRP003810 Other EXPression AloNg Development and Evolution EXPANDE project aims to identify gene expression profiles expanded during embryogenesis and evolution. In brief taking advantages of Illumina sequencing RNAseq profiles of early to late embryos of 8 chordate species were identified with biological replicates two or more biological replicates.     mRNA extracted from pooled embryos of 50 individuals Dr 72h 1 SAMD00028145   sample name:Dr 72h 1|strain:Riken WT Wild Type|tissue type:whole embryo|dev stage:72h Protruding mouth|genotype:wild type|phenotype:wild type|sex:male female and mixed                 Illumina HiSeq 2000 sequencing of SAMD00028145 DRX029554 Dr 72h 1 1 Total RNA QIAGEN RNeasy followed by TruSeq   RNA-Seq TRANSCRIPTOMIC other SINGLE ILLUMINA Illumina HiSeq 2000 <SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>100</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR> DRP003810 Illumina HiSeq 2000 sequencing of SAMD00028145       3897194500.0 38971945.0 DRR032748 0:100 1:0 A:1050989414;C:903225496;G:895783177;T:1047153493;N:42920 100 0     1050989414 903225496 895783177 1047153493 42920 DRX029554 DRS049953 DRA003460 UT-BS|Lab for embryology, Department of Biological Sciences, University of Tokyo UT-BS|Lab for embryology, Department of Biological Sciences, University of Tokyo 1 0.92211   0.09393   0.65486   0.45971   100   B   usable mapping rate illumina hiseq_era unknown other trueseq bulk unknown unknown   Japan 2017-09-20 Larval Larval Whole Organism All anatomical structures
81 81 DRR032743 DRX029549 DRS049948 DRP003810 PRJDB3785 EXPANDE project DRP003810 Other EXPression AloNg Development and Evolution EXPANDE project aims to identify gene expression profiles expanded during embryogenesis and evolution. In brief taking advantages of Illumina sequencing RNAseq profiles of early to late embryos of 8 chordate species were identified with biological replicates two or more biological replicates.     mRNA extracted from pooled embryos of 50 individuals Dr 5day 3 SAMD00028140   sample name:Dr 5day 3|strain:Riken WT Wild Type|tissue type:whole embryo|dev stage:5day|genotype:wild type|phenotype:wild type|sex:male female and mixed                 Illumina HiSeq 2000 sequencing of SAMD00028140 DRX029549 Dr 5day 3 1 Total RNA QIAGEN RNeasy followed by TruSeq   RNA-Seq TRANSCRIPTOMIC other SINGLE ILLUMINA Illumina HiSeq 2000 <SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>100</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR> DRP003810 Illumina HiSeq 2000 sequencing of SAMD00028140       3884716000.0 38847160.0 DRR032743 0:100 1:0 A:1040550584;C:905663425;G:904247323;T:1034215019;N:39649 100 0     1040550584 905663425 904247323 1034215019 39649 DRX029549 DRS049948 DRA003460 UT-BS|Lab for embryology, Department of Biological Sciences, University of Tokyo UT-BS|Lab for embryology, Department of Biological Sciences, University of Tokyo 1 0.92219   0.08287   0.65863   0.47377   100   B   usable mapping rate illumina hiseq_era unknown other trueseq bulk unknown unknown   Japan 2017-09-20 Larval Larval Whole Organism All anatomical structures
82 82 DRR032742 DRX029548 DRS049947 DRP003810 PRJDB3785 EXPANDE project DRP003810 Other EXPression AloNg Development and Evolution EXPANDE project aims to identify gene expression profiles expanded during embryogenesis and evolution. In brief taking advantages of Illumina sequencing RNAseq profiles of early to late embryos of 8 chordate species were identified with biological replicates two or more biological replicates.     mRNA extracted from pooled embryos of 50 individuals Dr 5day 2 SAMD00028139   sample name:Dr 5day 2|strain:Riken WT Wild Type|tissue type:whole embryo|dev stage:5day|genotype:wild type|phenotype:wild type|sex:male female and mixed                 Illumina HiSeq 2000 sequencing of SAMD00028139 DRX029548 Dr 5day 2 1 Total RNA QIAGEN RNeasy followed by TruSeq   RNA-Seq TRANSCRIPTOMIC other SINGLE ILLUMINA Illumina HiSeq 2000 <SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>100</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR> DRP003810 Illumina HiSeq 2000 sequencing of SAMD00028139       3893708700.0 38937087.0 DRR032742 0:100 1:0 A:1050850168;C:899863467;G:897224776;T:1045729184;N:41105 100 0     1050850168 899863467 897224776 1045729184 41105 DRX029548 DRS049947 DRA003460 UT-BS|Lab for embryology, Department of Biological Sciences, University of Tokyo UT-BS|Lab for embryology, Department of Biological Sciences, University of Tokyo 1 0.91671   0.0991   0.65161   0.47454   100   B   usable mapping rate illumina hiseq_era unknown other trueseq bulk unknown unknown   Japan 2017-09-20 Larval Larval Whole Organism All anatomical structures
83 83 DRR032741 DRX029547 DRS049946 DRP003810 PRJDB3785 EXPANDE project DRP003810 Other EXPression AloNg Development and Evolution EXPANDE project aims to identify gene expression profiles expanded during embryogenesis and evolution. In brief taking advantages of Illumina sequencing RNAseq profiles of early to late embryos of 8 chordate species were identified with biological replicates two or more biological replicates.     mRNA extracted from pooled embryos of 50 individuals Dr 5day 1 SAMD00028138   sample name:Dr 5day 1|strain:Riken WT Wild Type|tissue type:whole embryo|dev stage:5day|genotype:wild type|phenotype:wild type|sex:male female and mixed                 Illumina HiSeq 2000 sequencing of SAMD00028138 DRX029547 Dr 5day 1 1 Total RNA QIAGEN RNeasy followed by TruSeq   RNA-Seq TRANSCRIPTOMIC other SINGLE ILLUMINA Illumina HiSeq 2000 <SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>100</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR> DRP003810 Illumina HiSeq 2000 sequencing of SAMD00028138       3807570600.0 38075706.0 DRR032741 0:100 1:0 A:1022590228;C:884655401;G:882546091;T:1017737883;N:40997 100 0     1022590228 884655401 882546091 1017737883 40997 DRX029547 DRS049946 DRA003460 UT-BS|Lab for embryology, Department of Biological Sciences, University of Tokyo UT-BS|Lab for embryology, Department of Biological Sciences, University of Tokyo 1 0.9182   0.09442   0.65525   0.46661   100   B   usable mapping rate illumina hiseq_era unknown other trueseq bulk unknown unknown   Japan 2017-09-20 Larval Larval Whole Organism All anatomical structures
7947 7947 ERR015564 ERX005933 ERS012706 ERP000263 PRJEB2208 Zebrafish gene three prime end pull down for genome annotation E-MTAB-308 Transcriptome Analysis         E MTAB 308:Zebrafish embryo 3 dpf 2 SAMEA898404 Wellcome Sanger Institute Age:3 days|Alias:E MTAB 308:Zebrafish embryo 3 dpf 2|Broker name:ArrayExpress|Description:Protocols: Zebrafish embyos or tissues were collected from a Tuebingen strain incross and grown at 28 C. Collected samples were snap frozen on dry ice and stored at 70 C Total RNA was extracted using Trizol Reagent Invitrogen following the manufacturer's instructions. Pellets were resuspended RNase free 10 mM Tris pH 7.5 and the RNA was quantified using a NanoDrop ND 1000 Spectrophotometer Axon Instruments.|DevelopmentalStage:embryo|INSDC center alias:SC|INSDC center name:Wellcome Sanger Institute|INSDC first public:2010 08 19T15:57:35Z|INSDC last update:2018 03 08T15:25:04Z|INSDC status:public|InitialTimePoint:fertilization|OrganismPart:whole organism|SRA accession:ERS012706|Sample Name:ERS012706|Sex:unknown sex|StrainOrLine:Tuebingen|Title:Danio rerio                 Illumina Genome Analyzer II paired end sequencing; Zebrafish gene 3 prime end pull down for genome annotation E MTAB 308:Illumina Genome Analyzer II sequencing of Zebrafish embryo 3 dpf three prime pull down paired end 250 to 300 bp insert Zebrafish embro 3 dpf mRNA three prime end Zebrafish gene three prime end pull down for genome annotation 20 ug of total RNA was fragmented using RNA Fragmentation Reagent Ambion for 5 minutes at 70 C and ethanol precipitated with glycogen and LiCl. RNA was annealed to the oligo stBPM1polyT22 biotin GGCCAGTCCTGGAGTTTTTTTTTTTTTTTTTTTTTTVN and bound to streptavidin magnetic beads. post washing by pull down on a magnet the bound RNA was reverse transcribed with SuperScript II Invitrogen and a second strand synthesised with DNA polymerase I Promega and RNase H NEB. post further washing the double strand cDNA was released from the beads with BpmI NEB. The cDNA was recovered with the QIAgen PCR Purification Kit and made into a standard Illumina library following the manufacturer's protocol with a fragment size of 250 to 300 bp. Experimental Factor: AGE:3 d|Experimental Factor: DEVELOPMENTAL STAGE:embryo|Experimental Factor: ORGANISM PART:whole organism|Experimental Factor: SEX:unknown sex RNA-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina Genome Analyzer II   ERP000263 Illumina Genome Analyzer II paired end sequencing; Zebrafish gene three prime end pull down for genome annotation ENA FIRST PUBLIC:2010 08 19|ENA LAST UPDATE:2018 11 16 3444_3.srf srf 1572215648.0 10343524.0 E MTAB 308:Illumina Genome Analyzer II sequencing of Zebrafish embryo 3 dpf three prime pull down paired end 250 to 300 bp insert 0:76 1:76 A:395189954;C:374281517;G:356666372;T:420372888;N:25704917 76 76     395189954 374281517 356666372 420372888 25704917 ERX005933 ERS012706 ERA010603 SC|Wellcome Trust Sanger Institute SC|Wellcome Trust Sanger Institute 2 0.96337 0.96626 0.12558 0.12974 0.7824 0.79086 0.40731 0.41802 76 76 B B biological fallback assumption illumina early_illumina 3prime other unknown bulk unknown unknown   United Kingdom 2010-08-19 Larval Larval Whole Organism All anatomical structures
7951 7951 ERR015565 ERX005929 ERS012704 ERP000263 PRJEB2208 Zebrafish gene three prime end pull down for genome annotation E-MTAB-308 Transcriptome Analysis         E MTAB 308:Zebrafish embryo 5 dpf 2 SAMEA980815 SC Age:5 days|Alias:E MTAB 308:Zebrafish embryo 5 dpf 2|Broker name:ArrayExpress|Description:Protocols: Zebrafish embyos or tissues were collected from a Tuebingen strain incross and grown at 28 C. Collected samples were snap frozen on dry ice and stored at 70 C Total RNA was extracted using Trizol Reagent Invitrogen following the manufacturer's instructions. Pellets were resuspended RNase free 10 mM Tris pH 7.5 and the RNA was quantified using a NanoDrop ND 1000 Spectrophotometer Axon Instruments.|DevelopmentalStage:embryo|INSDC center name:SC|INSDC first public:2010 08 19T15:57:35Z|INSDC last update:2018 03 08T15:25:04Z|INSDC status:public|InitialTimePoint:fertilization|OrganismPart:whole organism|SRA accession:ERS012704|Sample Name:ERS012704|Sex:unknown sex|StrainOrLine:Tuebingen|Title:Danio rerio                 Illumina Genome Analyzer II paired end sequencing; Zebrafish gene 3 prime end pull down for genome annotation E MTAB 308:Illumina Genome Analyzer II sequencing of Zebrafish embryo 5 dpf three prime pull down paired end 250 to 300 bp insert Zebrafish embro 5 dpf mRNA three prime end Zebrafish gene three prime end pull down for genome annotation 20 ug of total RNA was fragmented using RNA Fragmentation Reagent Ambion for 5 minutes at 70 C and ethanol precipitated with glycogen and LiCl. RNA was annealed to the oligo stBPM1polyT22 biotin GGCCAGTCCTGGAGTTTTTTTTTTTTTTTTTTTTTTVN and bound to streptavidin magnetic beads. post washing by pull down on a magnet the bound RNA was reverse transcribed with SuperScript II Invitrogen and a second strand synthesised with DNA polymerase I Promega and RNase H NEB. post further washing the double strand cDNA was released from the beads with BpmI NEB. The cDNA was recovered with the QIAgen PCR Purification Kit and made into a standard Illumina library following the manufacturer's protocol with a fragment size of 250 to 300 bp. Experimental Factor: AGE:5 d|Experimental Factor: DEVELOPMENTAL STAGE:embryo|Experimental Factor: ORGANISM PART:whole organism|Experimental Factor: SEX:unknown sex RNA-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina Genome Analyzer II   ERP000263 Illumina Genome Analyzer II paired end sequencing; Zebrafish gene three prime end pull down for genome annotation ENA FIRST PUBLIC:2010 08 19|ENA LAST UPDATE:2018 11 16 3444_5.srf srf 1399629832.0 9208091.0 E MTAB 308:Illumina Genome Analyzer II sequencing of Zebrafish embryo 5 dpf three prime pull down paired end 250 to 300 bp insert 0:76 1:76 A:389123080;C:293319124;G:279736391;T:414876632;N:22574605 76 76     389123080 293319124 279736391 414876632 22574605 ERX005929 ERS012704 ERA010603 SC|Wellcome Trust Sanger Institute SC|Wellcome Trust Sanger Institute 2 0.92999 0.9332 0.45309 0.46162 0.72419 0.73919 0.49892 0.50512 76 76 B B biological fallback assumption illumina early_illumina 3prime other unknown bulk unknown unknown   United Kingdom 2010-08-19 Larval Larval Whole Organism All anatomical structures
34115 34115 SRR31230228 SRX26610547 SRS23109428 SRP543263 PRJNA1182301 Zebrafish Transcriptome data PRJNA1182301 Other Improving symptoms of light induced anxiety like insomnia in a zebrafish model.         TF L1   breed:Danio rerio|age:3 days|collection date:2022 10|geo loc name:not collected|sex:mixed|tissue:whole body|treatment:TF|Replicate:1|BioSampleModel:Model organism or animal                 Zebrafish Transcriptome data TF L1 TF L1 TF     RNA-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 1500   SRP543263     TF_L1_1.fq.gz TF_L1_2.fq.gz fastq fastq 6687564300.0 22291881.0 TF L1 1.fq.gz 0:150 1:150 A:1864117630;C:1488483222;G:1497677628;T:1837006551;N:279269 150 150     1864117630 1488483222 1497677628 1837006551 279269 SRX26610547 SRS23109428 SRA2005827 Shanghai Ocean University|College of Food Science &amp; Technology Shanghai Ocean University                       B B biological fallback assumption illumina hiseq_era unknown other unknown bulk unknown unknown   China 2024-11-05 Larval Larval Trunk Surface Structure
34116 34116 SRR31230229 SRX26610546 SRS23109427 SRP543263 PRJNA1182301 Zebrafish Transcriptome data PRJNA1182301 Other Improving symptoms of light induced anxiety like insomnia in a zebrafish model.         HP L3   breed:Danio rerio|age:3 days|collection date:2022 10|geo loc name:not collected|sex:mixed|tissue:whole body|treatment:HP|Replicate:3|BioSampleModel:Model organism or animal                 Zebrafish Transcriptome data HP L3 HP L3 HP     RNA-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 1500   SRP543263     HP_L3_1.fq.gz HP_L3_2.fq.gz fastq fastq 6481773600.0 21605912.0 HP L3 1.fq.gz 0:150 1:150 A:1822457830;C:1426224217;G:1433346356;T:1799447632;N:297565 150 150     1822457830 1426224217 1433346356 1799447632 297565 SRX26610546 SRS23109427 SRA2005827 Shanghai Ocean University|College of Food Science &amp; Technology Shanghai Ocean University                       B B biological fallback assumption illumina hiseq_era unknown other unknown bulk unknown unknown   China 2024-11-05 Larval Larval Trunk Surface Structure
34117 34117 SRR31230230 SRX26610545 SRS23109426 SRP543263 PRJNA1182301 Zebrafish Transcriptome data PRJNA1182301 Other Improving symptoms of light induced anxiety like insomnia in a zebrafish model.         HP L2   breed:Danio rerio|age:3 days|collection date:2022 10|geo loc name:not collected|sex:mixed|tissue:whole body|treatment:HP|Replicate:2|BioSampleModel:Model organism or animal                 Zebrafish Transcriptome data HP L2 HP L2 HP     RNA-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 1500   SRP543263     HP_L2_1.fq.gz HP_L2_2.fq.gz fastq fastq 6483969900.0 21613233.0 HP L2 1.fq.gz 0:150 1:150 A:1858850647;C:1400634811;G:1393756557;T:1830424227;N:303658 150 150     1858850647 1400634811 1393756557 1830424227 303658 SRX26610545 SRS23109426 SRA2005827 Shanghai Ocean University|College of Food Science &amp; Technology Shanghai Ocean University                       B B biological fallback assumption illumina hiseq_era unknown other unknown bulk unknown unknown   China 2024-11-05 Larval Larval Trunk Surface Structure
34118 34118 SRR31230231 SRX26610544 SRS23109422 SRP543263 PRJNA1182301 Zebrafish Transcriptome data PRJNA1182301 Other Improving symptoms of light induced anxiety like insomnia in a zebrafish model.         HP L1   breed:Danio rerio|age:3 days|collection date:2022 10|geo loc name:not collected|sex:mixed|tissue:whole body|treatment:HP|Replicate:1|BioSampleModel:Model organism or animal                 Zebrafish Transcriptome data HP L1 HP L1 HP     RNA-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 1500   SRP543263     HP_L1_1.fq.gz HP_L1_2.fq.gz fastq fastq 6270987900.0 20903293.0 HP L1 1.fq.gz 0:150 1:150 A:1761336291;C:1386332040;G:1384745780;T:1738282105;N:291684 150 150     1761336291 1386332040 1384745780 1738282105 291684 SRX26610544 SRS23109422 SRA2005827 Shanghai Ocean University|College of Food Science &amp; Technology Shanghai Ocean University                       B B biological fallback assumption illumina hiseq_era unknown other unknown bulk unknown unknown   China 2024-11-05 Larval Larval Trunk Surface Structure
34119 34119 SRR31230232 SRX26610543 SRS23109425 SRP543263 PRJNA1182301 Zebrafish Transcriptome data PRJNA1182301 Other Improving symptoms of light induced anxiety like insomnia in a zebrafish model.         MT L3   breed:Danio rerio|age:3 days|collection date:2022 10|geo loc name:not collected|sex:mixed|tissue:whole body|treatment:MT|Replicate:3|BioSampleModel:Model organism or animal                 Zebrafish Transcriptome data MT L3 MT L3 MT     RNA-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 1500   SRP543263     MT_L3_1.fq.gz MT_L3_2.fq.gz fastq fastq 6641395500.0 22137985.0 MT L3 1.fq.gz 0:150 1:150 A:1870037050;C:1459746735;G:1464312013;T:1846983464;N:316238 150 150     1870037050 1459746735 1464312013 1846983464 316238 SRX26610543 SRS23109425 SRA2005827 Shanghai Ocean University|College of Food Science &amp; Technology Shanghai Ocean University                       B B biological fallback assumption illumina hiseq_era unknown other unknown bulk unknown unknown   China 2024-11-05 Larval Larval Trunk Surface Structure
34120 34120 SRR31230233 SRX26610542 SRS23109421 SRP543263 PRJNA1182301 Zebrafish Transcriptome data PRJNA1182301 Other Improving symptoms of light induced anxiety like insomnia in a zebrafish model.         MT L2   breed:Danio rerio|age:3 days|collection date:2022 10|geo loc name:not collected|sex:mixed|tissue:whole body|treatment:MT|Replicate:2|BioSampleModel:Model organism or animal                 Zebrafish Transcriptome data MT L2 MT L2 MT     RNA-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 1500   SRP543263     MT_L2_1.fq.gz MT_L2_2.fq.gz fastq fastq 6735230400.0 22450768.0 MT L2 1.fq.gz 0:150 1:150 A:1884876161;C:1494102137;G:1499635722;T:1856320830;N:295550 150 150     1884876161 1494102137 1499635722 1856320830 295550 SRX26610542 SRS23109421 SRA2005827 Shanghai Ocean University|College of Food Science &amp; Technology Shanghai Ocean University                       B B biological fallback assumption illumina hiseq_era unknown other unknown bulk unknown unknown   China 2024-11-05 Larval Larval Trunk Surface Structure
34121 34121 SRR31230234 SRX26610541 SRS23109424 SRP543263 PRJNA1182301 Zebrafish Transcriptome data PRJNA1182301 Other Improving symptoms of light induced anxiety like insomnia in a zebrafish model.         MT L1   breed:Danio rerio|age:3 days|collection date:2022 10|geo loc name:not collected|sex:mixed|tissue:whole body|treatment:MT|Replicate:1|BioSampleModel:Model organism or animal                 Zebrafish Transcriptome data MT L1 MT L1 MT     RNA-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 1500   SRP543263     MT_L1_1.fq.gz MT_L1_2.fq.gz fastq fastq 6382458300.0 21274861.0 MT L1 1.fq.gz 0:150 1:150 A:1811407290;C:1396361259;G:1388659914;T:1785732773;N:297064 150 150     1811407290 1396361259 1388659914 1785732773 297064 SRX26610541 SRS23109424 SRA2005827 Shanghai Ocean University|College of Food Science &amp; Technology Shanghai Ocean University                       B B biological fallback assumption illumina hiseq_era unknown other unknown bulk unknown unknown   China 2024-11-05 Larval Larval Trunk Surface Structure
34122 34122 SRR31230235 SRX26610540 SRS23109423 SRP543263 PRJNA1182301 Zebrafish Transcriptome data PRJNA1182301 Other Improving symptoms of light induced anxiety like insomnia in a zebrafish model.         NC L3   breed:Danio rerio|age:3 days|collection date:2022 10|geo loc name:not collected|sex:mixed|tissue:whole body|treatment:Control|Replicate:3|BioSampleModel:Model organism or animal                 Zebrafish Transcriptome data NC L3 NC L3 control     RNA-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 1500   SRP543263     NC_L3_1.fq.gz NC_L3_2.fq.gz fastq fastq 6815267400.0 22717558.0 NC L3 1.fq.gz 0:150 1:150 A:1909187573;C:1516756161;G:1522759558;T:1866449373;N:114735 150 150     1909187573 1516756161 1522759558 1866449373 114735 SRX26610540 SRS23109423 SRA2005827 Shanghai Ocean University|College of Food Science &amp; Technology Shanghai Ocean University                       B B biological fallback assumption illumina hiseq_era unknown other unknown bulk unknown unknown   China 2024-11-05 Larval Larval Trunk Surface Structure
34123 34123 SRR31230236 SRX26610539 SRS23109419 SRP543263 PRJNA1182301 Zebrafish Transcriptome data PRJNA1182301 Other Improving symptoms of light induced anxiety like insomnia in a zebrafish model.         TF L3   breed:Danio rerio|age:3 days|collection date:2022 10|geo loc name:not collected|sex:mixed|tissue:whole body|treatment:TF|Replicate:3|BioSampleModel:Model organism or animal                 Zebrafish Transcriptome data TF L3 TF L3 TF     RNA-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 1500   SRP543263     TF_L3_1.fq.gz TF_L3_2.fq.gz fastq fastq 6547426200.0 21824754.0 TF L3 1.fq.gz 0:150 1:150 A:1829107582;C:1450999468;G:1461503884;T:1805507445;N:307821 150 150     1829107582 1450999468 1461503884 1805507445 307821 SRX26610539 SRS23109419 SRA2005827 Shanghai Ocean University|College of Food Science &amp; Technology Shanghai Ocean University                       B B biological fallback assumption illumina hiseq_era unknown other unknown bulk unknown unknown   China 2024-11-05 Larval Larval Trunk Surface Structure
34124 34124 SRR31230237 SRX26610538 SRS23109420 SRP543263 PRJNA1182301 Zebrafish Transcriptome data PRJNA1182301 Other Improving symptoms of light induced anxiety like insomnia in a zebrafish model.         TF L2   breed:Danio rerio|age:3 days|collection date:2022 10|geo loc name:not collected|sex:mixed|tissue:whole body|treatment:TF|Replicate:2|BioSampleModel:Model organism or animal                 Zebrafish Transcriptome data TF L2 TF L2 TF     RNA-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 1500   SRP543263     TF_L2_1.fq.gz TF_L2_2.fq.gz fastq fastq 6327866400.0 21092888.0 TF L2 1.fq.gz 0:150 1:150 A:1792246492;C:1386064311;G:1383369731;T:1765884262;N:301604 150 150     1792246492 1386064311 1383369731 1765884262 301604 SRX26610538 SRS23109420 SRA2005827 Shanghai Ocean University|College of Food Science &amp; Technology Shanghai Ocean University                       B B biological fallback assumption illumina hiseq_era unknown other unknown bulk unknown unknown   China 2024-11-05 Larval Larval Trunk Surface Structure
34125 34125 SRR31230238 SRX26610537 SRS23109418 SRP543263 PRJNA1182301 Zebrafish Transcriptome data PRJNA1182301 Other Improving symptoms of light induced anxiety like insomnia in a zebrafish model.         NC L2   breed:Danio rerio|age:3 days|collection date:2022 10|geo loc name:not collected|sex:mixed|tissue:whole body|treatment:Control|Replicate:2|BioSampleModel:Model organism or animal                 Zebrafish Transcriptome data NC L2 NC L2 control     RNA-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 1500   SRP543263     NC_L2_1.fq.gz NC_L2_2.fq.gz fastq fastq 6779235000.0 22597450.0 NC L2 1.fq.gz 0:150 1:150 A:1908783350;C:1490731242;G:1502686284;T:1876708170;N:325954 150 150     1908783350 1490731242 1502686284 1876708170 325954 SRX26610537 SRS23109418 SRA2005827 Shanghai Ocean University|College of Food Science &amp; Technology Shanghai Ocean University                       B B biological fallback assumption illumina hiseq_era unknown other unknown bulk unknown unknown   China 2024-11-05 Larval Larval Trunk Surface Structure
34126 34126 SRR31230239 SRX26610536 SRS23109417 SRP543263 PRJNA1182301 Zebrafish Transcriptome data PRJNA1182301 Other Improving symptoms of light induced anxiety like insomnia in a zebrafish model.         NC L1   breed:Danio rerio|age:3 days|collection date:2022 10|geo loc name:not collected|sex:mixed|tissue:whole body|treatment:Control|Replicate:1|BioSampleModel:Model organism or animal                 Zebrafish Transcriptome data NC L1 NC L1 control     RNA-Seq TRANSCRIPTOMIC other PAIRED ILLUMINA Illumina HiSeq 1500   SRP543263     NC_L1_1.fq.gz NC_L1_2.fq.gz fastq fastq 6318379500.0 21061265.0 NC L1 1.fq.gz 0:150 1:150 A:1785873867;C:1385813719;G:1388477232;T:1757923663;N:291019 150 150     1785873867 1385813719 1388477232 1757923663 291019 SRX26610536 SRS23109417 SRA2005827 Shanghai Ocean University|College of Food Science &amp; Technology Shanghai Ocean University                       B B biological fallback assumption illumina hiseq_era unknown other unknown bulk unknown unknown   China 2024-11-05 Larval Larval Trunk Surface Structure
34712 34712 SRR32232924 SRX27573024 SRS23984677 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 05H GSM8775334   source name:6 dpf head|tissue:6 dpf head|well:P1 H05|rtindex:TTCGCCTCCA|genotype:GAL4 replicate2|geo loc name:missing|collection date:missing Well P1 05H Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 H05|rtindex:TTCGCCTCCA|genotype:GAL4 replicate2 GSM8775334 GSM8775334: Well P1 05H; Danio rerio; OTHER GSM8775334 r1 GSM8775334 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-05H.R2.fastq.gz P1-05H_S40_I1_001.fastq.gz P1-05H.R1.fastq.gz fastq fastq fastq 828.0 9.0 GSM8775334 r1 0:34 1:48 2:10 A:210;C:74;G:47;T:101;N:0 34 48 10   210 74 47 101 0 SRX27573024 SRS23984677 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34713 34713 SRR32232925 SRX27573023 SRS23984676 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 05G GSM8775333   source name:6 dpf head|tissue:6 dpf head|well:P1 G05|rtindex:TTACCTCGAC|genotype:GAL4 replicate2|geo loc name:missing|collection date:missing Well P1 05G Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 G05|rtindex:TTACCTCGAC|genotype:GAL4 replicate2 GSM8775333 GSM8775333: Well P1 05G; Danio rerio; OTHER GSM8775333 r1 GSM8775333 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-05G.R1.fastq.gz P1-05G.R2.fastq.gz P1-05G_S39_I1_001.fastq.gz fastq fastq fastq 3345856.0 36368.0 GSM8775333 r1 0:34 1:48 2:10 A:602824;C:246794;G:422064;T:471661;N:2321 34 48 10   602824 246794 422064 471661 2321 SRX27573023 SRS23984676 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34714 34714 SRR32232926 SRX27573022 SRS23984675 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 05F GSM8775332   source name:6 dpf head|tissue:6 dpf head|well:P1 F05|rtindex:CCATTATCTA|genotype:GAL4 replicate2|geo loc name:missing|collection date:missing Well P1 05F Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 F05|rtindex:CCATTATCTA|genotype:GAL4 replicate2 GSM8775332 GSM8775332: Well P1 05F; Danio rerio; OTHER GSM8775332 r1 GSM8775332 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-05F.R1.fastq.gz P1-05F.R2.fastq.gz P1-05F_S38_I1_001.fastq.gz fastq fastq fastq 143353940.0 1558195.0 GSM8775332 r1 0:34 1:48 2:10 A:32466011;C:9391164;G:14603239;T:18226855;N:106091 34 48 10   32466011 9391164 14603239 18226855 106091 SRX27573022 SRS23984675 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34715 34715 SRR32232927 SRX27573021 SRS23984674 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 05E GSM8775331   source name:6 dpf head|tissue:6 dpf head|well:P1 E05|rtindex:CTAGTAGTCT|genotype:GAL4 replicate2|geo loc name:missing|collection date:missing Well P1 05E Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 E05|rtindex:CTAGTAGTCT|genotype:GAL4 replicate2 GSM8775331 GSM8775331: Well P1 05E; Danio rerio; OTHER GSM8775331 r1 GSM8775331 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-05E.R1.fastq.gz P1-05E.R2.fastq.gz P1-05E_S37_I1_001.fastq.gz fastq fastq fastq 45118824.0 490422.0 GSM8775331 r1 0:34 1:48 2:10 A:7745921;C:3203071;G:7199342;T:5360465;N:31457 34 48 10   7745921 3203071 7199342 5360465 31457 SRX27573021 SRS23984674 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34716 34716 SRR32232928 SRX27573020 SRS23984673 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 05D GSM8775330   source name:6 dpf head|tissue:6 dpf head|well:P1 D05|rtindex:AGAGAAGGTT|genotype:GAL4 replicate2|geo loc name:missing|collection date:missing Well P1 05D Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 D05|rtindex:AGAGAAGGTT|genotype:GAL4 replicate2 GSM8775330 GSM8775330: Well P1 05D; Danio rerio; OTHER GSM8775330 r1 GSM8775330 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-05D.R1.fastq.gz P1-05D.R2.fastq.gz P1-05D_S36_I1_001.fastq.gz fastq fastq fastq 194869248.0 2118144.0 GSM8775330 r1 0:34 1:48 2:10 A:44069791;C:12867012;G:18935172;T:25657047;N:141890 34 48 10   44069791 12867012 18935172 25657047 141890 SRX27573020 SRS23984673 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34717 34717 SRR32232929 SRX27573019 SRS23984672 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 05C GSM8775329   source name:6 dpf head|tissue:6 dpf head|well:P1 C05|rtindex:TAGAGTAATA|genotype:GAL4 replicate2|geo loc name:missing|collection date:missing Well P1 05C Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 C05|rtindex:TAGAGTAATA|genotype:GAL4 replicate2 GSM8775329 GSM8775329: Well P1 05C; Danio rerio; OTHER GSM8775329 r1 GSM8775329 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-05C.R1.fastq.gz P1-05C.R2.fastq.gz P1-05C_S35_I1_001.fastq.gz fastq fastq fastq 47118352.0 512156.0 GSM8775329 r1 0:34 1:48 2:10 A:9098221;C:3194198;G:6516993;T:5738665;N:35411 34 48 10   9098221 3194198 6516993 5738665 35411 SRX27573019 SRS23984672 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34718 34718 SRR32232930 SRX27573018 SRS23984671 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 05B GSM8775328   source name:6 dpf head|tissue:6 dpf head|well:P1 B05|rtindex:CTTGCGCCGC|genotype:GAL4 replicate2|geo loc name:missing|collection date:missing Well P1 05B Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 B05|rtindex:CTTGCGCCGC|genotype:GAL4 replicate2 GSM8775328 GSM8775328: Well P1 05B; Danio rerio; OTHER GSM8775328 r1 GSM8775328 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-05B.R1.fastq.gz P1-05B.R2.fastq.gz P1-05B_S34_I1_001.fastq.gz fastq fastq fastq 664471748.0 7222519.0 GSM8775328 r1 0:34 1:48 2:10 A:146424611;C:46287832;G:64801496;T:88677718;N:489255 34 48 10   146424611 46287832 64801496 88677718 489255 SRX27573018 SRS23984671 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34719 34719 SRR32232931 SRX27573017 SRS23984670 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 05A GSM8775327   source name:6 dpf head|tissue:6 dpf head|well:P1 A05|rtindex:CCGAGAATCC|genotype:GAL4 replicate2|geo loc name:missing|collection date:missing Well P1 05A Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 A05|rtindex:CCGAGAATCC|genotype:GAL4 replicate2 GSM8775327 GSM8775327: Well P1 05A; Danio rerio; OTHER GSM8775327 r1 GSM8775327 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-05A.R1.fastq.gz P1-05A.R2.fastq.gz P1-05A_S33_I1_001.fastq.gz fastq fastq fastq 33378152.0 362806.0 GSM8775327 r1 0:34 1:48 2:10 A:5712677;C:2316258;G:5388122;T:3975276;N:22355 34 48 10   5712677 2316258 5388122 3975276 22355 SRX27573017 SRS23984670 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34720 34720 SRR32232932 SRX27573016 SRS23984669 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 12H GSM8775389   source name:6 dpf head|tissue:6 dpf head|well:P1 H12|rtindex:TAACTGGTTA|genotype:UAS replicate3|geo loc name:missing|collection date:missing Well P1 12H Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 H12|rtindex:TAACTGGTTA|genotype:UAS replicate3 GSM8775389 GSM8775389: Well P1 12H; Danio rerio; OTHER GSM8775389 r1 GSM8775389 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-12H.R1.fastq.gz P1-12H.R2.fastq.gz P1-12H_S96_I1_001.fastq.gz fastq fastq fastq 368.0 4.0 GSM8775389 r1 0:34 1:48 2:10 A:70;C:25;G:61;T:36;N:0 34 48 10   70 25 61 36 0 SRX27573016 SRS23984669 SRA2070568 UMass Chan UMass Chan                       T   under 1.2% mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34721 34721 SRR32232933 SRX27573015 SRS23984668 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 12G GSM8775388   source name:6 dpf head|tissue:6 dpf head|well:P1 G12|rtindex:GGTACCGGCA|genotype:UAS replicate3|geo loc name:missing|collection date:missing Well P1 12G Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 G12|rtindex:GGTACCGGCA|genotype:UAS replicate3 GSM8775388 GSM8775388: Well P1 12G; Danio rerio; OTHER GSM8775388 r1 GSM8775388 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-12G.R1.fastq.gz P1-12G.R2.fastq.gz P1-12G_S95_I1_001.fastq.gz fastq fastq fastq 11776.0 128.0 GSM8775388 r1 0:34 1:48 2:10 A:2531;C:932;G:1152;T:1529;N:0 34 48 10   2531 932 1152 1529 0 SRX27573015 SRS23984668 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34722 34722 SRR32232934 SRX27573014 SRS23984666 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 12F GSM8775387   source name:6 dpf head|tissue:6 dpf head|well:P1 F12|rtindex:CATTCGACGG|genotype:UAS replicate3|geo loc name:missing|collection date:missing Well P1 12F Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 F12|rtindex:CATTCGACGG|genotype:UAS replicate3 GSM8775387 GSM8775387: Well P1 12F; Danio rerio; OTHER GSM8775387 r1 GSM8775387 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-12F.R1.fastq.gz P1-12F.R2.fastq.gz P1-12F_S94_I1_001.fastq.gz fastq fastq fastq 1012.0 11.0 GSM8775387 r1 0:34 1:48 2:10 A:192;C:67;G:130;T:139;N:0 34 48 10   192 67 130 139 0 SRX27573014 SRS23984666 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34723 34723 SRR32232935 SRX27573013 SRS23984667 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 12E GSM8775386   source name:6 dpf head|tissue:6 dpf head|well:P1 E12|rtindex:AACCATTGGA|genotype:UAS replicate3|geo loc name:missing|collection date:missing Well P1 12E Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 E12|rtindex:AACCATTGGA|genotype:UAS replicate3 GSM8775386 GSM8775386: Well P1 12E; Danio rerio; OTHER GSM8775386 r1 GSM8775386 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-12E.R1.fastq.gz P1-12E.R2.fastq.gz P1-12E_S93_I1_001.fastq.gz fastq fastq fastq 512348.0 5569.0 GSM8775386 r1 0:34 1:48 2:10 A:84224;C:34420;G:85992;T:62126;N:550 34 48 10   84224 34420 85992 62126 550 SRX27573013 SRS23984667 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34724 34724 SRR32232936 SRX27573012 SRS23984665 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 12D GSM8775385   source name:6 dpf head|tissue:6 dpf head|well:P1 D12|rtindex:CGAACGCCGG|genotype:UAS replicate3|geo loc name:missing|collection date:missing Well P1 12D Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 D12|rtindex:CGAACGCCGG|genotype:UAS replicate3 GSM8775385 GSM8775385: Well P1 12D; Danio rerio; OTHER GSM8775385 r1 GSM8775385 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-12D.R1.fastq.gz P1-12D.R2.fastq.gz P1-12D_S92_I1_001.fastq.gz fastq fastq fastq 14633428.0 159059.0 GSM8775385 r1 0:34 1:48 2:10 A:2133794;C:938571;G:2850027;T:1703116;N:9324 34 48 10   2133794 938571 2850027 1703116 9324 SRX27573012 SRS23984665 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34725 34725 SRR32232937 SRX27573011 SRS23984664 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 12C GSM8775384   source name:6 dpf head|tissue:6 dpf head|well:P1 C12|rtindex:GACCTTGATA|genotype:UAS replicate3|geo loc name:missing|collection date:missing Well P1 12C Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 C12|rtindex:GACCTTGATA|genotype:UAS replicate3 GSM8775384 GSM8775384: Well P1 12C; Danio rerio; OTHER GSM8775384 r1 GSM8775384 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-12C.R1.fastq.gz P1-12C.R2.fastq.gz P1-12C_S91_I1_001.fastq.gz fastq fastq fastq 24830524.0 269897.0 GSM8775384 r1 0:34 1:48 2:10 A:3753240;C:1715145;G:4579242;T:2889684;N:17745 34 48 10   3753240 1715145 4579242 2889684 17745 SRX27573011 SRS23984664 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34726 34726 SRR32232938 SRX27573010 SRS23984663 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 12B GSM8775383   source name:6 dpf head|tissue:6 dpf head|well:P1 B12|rtindex:TGGTCAGCCA|genotype:UAS replicate3|geo loc name:missing|collection date:missing Well P1 12B Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 B12|rtindex:TGGTCAGCCA|genotype:UAS replicate3 GSM8775383 GSM8775383: Well P1 12B; Danio rerio; OTHER GSM8775383 r1 GSM8775383 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-12B.R1.fastq.gz P1-12B.R2.fastq.gz P1-12B_S90_I1_001.fastq.gz fastq fastq fastq 22899628.0 248909.0 GSM8775383 r1 0:34 1:48 2:10 A:3385725;C:1544608;G:4301274;T:2699465;N:16560 34 48 10   3385725 1544608 4301274 2699465 16560 SRX27573010 SRS23984663 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34727 34727 SRR32232939 SRX27573009 SRS23984662 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 08H GSM8775358   source name:6 dpf head|tissue:6 dpf head|well:P1 H08|rtindex:AGCTCTCCTC|genotype:UAS replicate2|geo loc name:missing|collection date:missing Well P1 08H Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 H08|rtindex:AGCTCTCCTC|genotype:UAS replicate2 GSM8775358 GSM8775358: Well P1 08H; Danio rerio; OTHER GSM8775358 r1 GSM8775358 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-08H.R1.fastq.gz P1-08H.R2.fastq.gz P1-08H_S64_I1_001.fastq.gz fastq fastq fastq 552.0 6.0 GSM8775358 r1 0:34 1:48 2:10 A:114;C:44;G:65;T:65;N:0 34 48 10   114 44 65 65 0 SRX27573009 SRS23984662 SRA2070568 UMass Chan UMass Chan                       T   under 1.2% mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34728 34728 SRR32232940 SRX27573008 SRS23984661 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 08G GSM8775357   source name:6 dpf head|tissue:6 dpf head|well:P1 G08|rtindex:CGGTCAAGAA|genotype:UAS replicate2|geo loc name:missing|collection date:missing Well P1 08G Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 G08|rtindex:CGGTCAAGAA|genotype:UAS replicate2 GSM8775357 GSM8775357: Well P1 08G; Danio rerio; OTHER GSM8775357 r1 GSM8775357 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-08G.R1.fastq.gz P1-08G.R2.fastq.gz P1-08G_S63_I1_001.fastq.gz fastq fastq fastq 48348484.0 525527.0 GSM8775357 r1 0:34 1:48 2:10 A:8706380;C:3262708;G:7330788;T:5889373;N:36047 34 48 10   8706380 3262708 7330788 5889373 36047 SRX27573008 SRS23984661 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34729 34729 SRR32232941 SRX27573007 SRS23984660 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 08F GSM8775356   source name:6 dpf head|tissue:6 dpf head|well:P1 F08|rtindex:GATCGCTTCT|genotype:UAS replicate2|geo loc name:missing|collection date:missing Well P1 08F Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 F08|rtindex:GATCGCTTCT|genotype:UAS replicate2 GSM8775356 GSM8775356: Well P1 08F; Danio rerio; OTHER GSM8775356 r1 GSM8775356 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-08F.R1.fastq.gz P1-08F.R2.fastq.gz P1-08F_S62_I1_001.fastq.gz fastq fastq fastq 55386116.0 602023.0 GSM8775356 r1 0:34 1:48 2:10 A:10289406;C:3851867;G:8139616;T:6575750;N:40465 34 48 10   10289406 3851867 8139616 6575750 40465 SRX27573007 SRS23984660 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34730 34730 SRR32232942 SRX27573006 SRS23984659 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 08E GSM8775355   source name:6 dpf head|tissue:6 dpf head|well:P1 E08|rtindex:AACTAGGCGC|genotype:UAS replicate2|geo loc name:missing|collection date:missing Well P1 08E Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 E08|rtindex:AACTAGGCGC|genotype:UAS replicate2 GSM8775355 GSM8775355: Well P1 08E; Danio rerio; OTHER GSM8775355 r1 GSM8775355 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-08E.R1.fastq.gz P1-08E.R2.fastq.gz P1-08E_S61_I1_001.fastq.gz fastq fastq fastq 40465832.0 439846.0 GSM8775355 r1 0:34 1:48 2:10 A:6595819;C:2847582;G:6802285;T:4838399;N:28523 34 48 10   6595819 2847582 6802285 4838399 28523 SRX27573006 SRS23984659 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34731 34731 SRR32232943 SRX27573005 SRS23984658 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 08D GSM8775354   source name:6 dpf head|tissue:6 dpf head|well:P1 D08|rtindex:GGCTGAGCTC|genotype:UAS replicate2|geo loc name:missing|collection date:missing Well P1 08D Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 D08|rtindex:GGCTGAGCTC|genotype:UAS replicate2 GSM8775354 GSM8775354: Well P1 08D; Danio rerio; OTHER GSM8775354 r1 GSM8775354 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-08D.R1.fastq.gz P1-08D.R2.fastq.gz P1-08D_S60_I1_001.fastq.gz fastq fastq fastq 33437492.0 363451.0 GSM8775354 r1 0:34 1:48 2:10 A:4392257;C:1994475;G:7440459;T:3596074;N:22383 34 48 10   4392257 1994475 7440459 3596074 22383 SRX27573005 SRS23984658 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34732 34732 SRR32232944 SRX27573004 SRS23984656 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 08C GSM8775353   source name:6 dpf head|tissue:6 dpf head|well:P1 C08|rtindex:AGAACGTCTC|genotype:UAS replicate2|geo loc name:missing|collection date:missing Well P1 08C Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 C08|rtindex:AGAACGTCTC|genotype:UAS replicate2 GSM8775353 GSM8775353: Well P1 08C; Danio rerio; OTHER GSM8775353 r1 GSM8775353 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-08C.R1.fastq.gz P1-08C.R2.fastq.gz P1-08C_S59_I1_001.fastq.gz fastq fastq fastq 29029864.0 315542.0 GSM8775353 r1 0:34 1:48 2:10 A:4629476;C:2009722;G:5052427;T:3434235;N:20156 34 48 10   4629476 2009722 5052427 3434235 20156 SRX27573004 SRS23984656 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34733 34733 SRR32232945 SRX27573003 SRS23984657 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 08B GSM8775352   source name:6 dpf head|tissue:6 dpf head|well:P1 B08|rtindex:GCCATTCTCC|genotype:UAS replicate2|geo loc name:missing|collection date:missing Well P1 08B Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 B08|rtindex:GCCATTCTCC|genotype:UAS replicate2 GSM8775352 GSM8775352: Well P1 08B; Danio rerio; OTHER GSM8775352 r1 GSM8775352 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-08B.R1.fastq.gz P1-08B.R2.fastq.gz P1-08B_S58_I1_001.fastq.gz fastq fastq fastq 7182683524.0 78072647.0 GSM8775352 r1 0:34 1:48 2:10 A:1191235462;C:707748234;G:767219572;T:1075959763;N:5324025 34 48 10   1191235462 707748234 767219572 1075959763 5324025 SRX27573003 SRS23984657 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34734 34734 SRR32232946 SRX27573002 SRS23984655 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 08A GSM8775351   source name:6 dpf head|tissue:6 dpf head|well:P1 A08|rtindex:TGCGGACCTA|genotype:UAS replicate2|geo loc name:missing|collection date:missing Well P1 08A Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 A08|rtindex:TGCGGACCTA|genotype:UAS replicate2 GSM8775351 GSM8775351: Well P1 08A; Danio rerio; OTHER GSM8775351 r1 GSM8775351 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-08A.R1.fastq.gz P1-08A.R2.fastq.gz P1-08A_S57_I1_001.fastq.gz fastq fastq fastq 33057440.0 359320.0 GSM8775351 r1 0:34 1:48 2:10 A:5780741;C:2263389;G:5259112;T:3921095;N:23023 34 48 10   5780741 2263389 5259112 3921095 23023 SRX27573002 SRS23984655 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34735 34735 SRR32232947 SRX27573001 SRS23984654 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 04H GSM8775326   source name:6 dpf head|tissue:6 dpf head|well:P1 H04|rtindex:AGGTTAGCAT|genotype:UAS replicate1|geo loc name:missing|collection date:missing Well P1 04H Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 H04|rtindex:AGGTTAGCAT|genotype:UAS replicate1 GSM8775326 GSM8775326: Well P1 04H; Danio rerio; OTHER GSM8775326 r1 GSM8775326 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-04H.R1.fastq.gz P1-04H.R2.fastq.gz P1-04H_S32_I1_001.fastq.gz fastq fastq fastq 4048.0 44.0 GSM8775326 r1 0:34 1:48 2:10 A:877;C:264;G:476;T:495;N:0 34 48 10   877 264 476 495 0 SRX27573001 SRS23984654 SRA2070568 UMass Chan UMass Chan                       T   under 1.2% mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34736 34736 SRR32232948 SRX27573000 SRS23984653 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 04G GSM8775325   source name:6 dpf head|tissue:6 dpf head|well:P1 G04|rtindex:ACGAAGTCAA|genotype:UAS replicate1|geo loc name:missing|collection date:missing Well P1 04G Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 G04|rtindex:ACGAAGTCAA|genotype:UAS replicate1 GSM8775325 GSM8775325: Well P1 04G; Danio rerio; OTHER GSM8775325 r1 GSM8775325 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-04G.R1.fastq.gz P1-04G.R2.fastq.gz P1-04G_S31_I1_001.fastq.gz fastq fastq fastq 16717412.0 181711.0 GSM8775325 r1 0:34 1:48 2:10 A:2534077;C:1123957;G:3093004;T:1959215;N:11875 34 48 10   2534077 1123957 3093004 1959215 11875 SRX27573000 SRS23984653 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34737 34737 SRR32232949 SRX27572999 SRS23984652 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 04F GSM8775324   source name:6 dpf head|tissue:6 dpf head|well:P1 F04|rtindex:AATCGATAAT|genotype:UAS replicate1|geo loc name:missing|collection date:missing Well P1 04F Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 F04|rtindex:AATCGATAAT|genotype:UAS replicate1 GSM8775324 GSM8775324: Well P1 04F; Danio rerio; OTHER GSM8775324 r1 GSM8775324 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-04F.R1.fastq.gz P1-04F.R2.fastq.gz P1-04F_S30_I1_001.fastq.gz fastq fastq fastq 71229712.0 774236.0 GSM8775324 r1 0:34 1:48 2:10 A:13212435;C:4888655;G:10555214;T:8456988;N:50036 34 48 10   13212435 4888655 10555214 8456988 50036 SRX27572999 SRS23984652 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34738 34738 SRR32232950 SRX27572998 SRS23984651 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 04E GSM8775323   source name:6 dpf head|tissue:6 dpf head|well:P1 E04|rtindex:TAGCCGTCAT|genotype:UAS replicate1|geo loc name:missing|collection date:missing Well P1 04E Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 E04|rtindex:TAGCCGTCAT|genotype:UAS replicate1 GSM8775323 GSM8775323: Well P1 04E; Danio rerio; OTHER GSM8775323 r1 GSM8775323 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-04E.R1.fastq.gz P1-04E.R2.fastq.gz P1-04E_S29_I1_001.fastq.gz fastq fastq fastq 381955388.0 4151689.0 GSM8775323 r1 0:34 1:48 2:10 A:87514409;C:25262710;G:36461600;T:49768768;N:273585 34 48 10   87514409 25262710 36461600 49768768 273585 SRX27572998 SRS23984651 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34739 34739 SRR32232951 SRX27572997 SRS23984649 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 04D GSM8775322   source name:6 dpf head|tissue:6 dpf head|well:P1 D04|rtindex:TCCGGCCTCG|genotype:UAS replicate1|geo loc name:missing|collection date:missing Well P1 04D Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 D04|rtindex:TCCGGCCTCG|genotype:UAS replicate1 GSM8775322 GSM8775322: Well P1 04D; Danio rerio; OTHER GSM8775322 r1 GSM8775322 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-04D.R1.fastq.gz P1-04D.R2.fastq.gz P1-04D_S28_I1_001.fastq.gz fastq fastq fastq 57109552.0 620756.0 GSM8775322 r1 0:34 1:48 2:10 A:11045441;C:3910161;G:7896770;T:6905332;N:38584 34 48 10   11045441 3910161 7896770 6905332 38584 SRX27572997 SRS23984649 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34740 34740 SRR32232952 SRX27572996 SRS23984650 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 04C GSM8775321   source name:6 dpf head|tissue:6 dpf head|well:P1 C04|rtindex:TTATCCGGAT|genotype:UAS replicate1|geo loc name:missing|collection date:missing Well P1 04C Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 C04|rtindex:TTATCCGGAT|genotype:UAS replicate1 GSM8775321 GSM8775321: Well P1 04C; Danio rerio; OTHER GSM8775321 r1 GSM8775321 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-04C.R1.fastq.gz P1-04C.R2.fastq.gz P1-04C_S27_I1_001.fastq.gz fastq fastq fastq 41102932.0 446771.0 GSM8775321 r1 0:34 1:48 2:10 A:7556055;C:2874427;G:6121487;T:4864586;N:28453 34 48 10   7556055 2874427 6121487 4864586 28453 SRX27572996 SRS23984650 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34741 34741 SRR32232953 SRX27572995 SRS23984648 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 04B GSM8775320   source name:6 dpf head|tissue:6 dpf head|well:P1 B04|rtindex:ACTCAATAGG|genotype:UAS replicate1|geo loc name:missing|collection date:missing Well P1 04B Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 B04|rtindex:ACTCAATAGG|genotype:UAS replicate1 GSM8775320 GSM8775320: Well P1 04B; Danio rerio; OTHER GSM8775320 r1 GSM8775320 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-04B.R1.fastq.gz P1-04B.R2.fastq.gz P1-04B_S26_I1_001.fastq.gz fastq fastq fastq 39908772.0 433791.0 GSM8775320 r1 0:34 1:48 2:10 A:7097865;C:2701376;G:6191071;T:4805009;N:26647 34 48 10   7097865 2701376 6191071 4805009 26647 SRX27572995 SRS23984648 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34742 34742 SRR32232954 SRX27572994 SRS23984647 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 04A GSM8775319   source name:6 dpf head|tissue:6 dpf head|well:P1 A04|rtindex:CTGATGGTCA|genotype:UAS replicate1|geo loc name:missing|collection date:missing Well P1 04A Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 A04|rtindex:CTGATGGTCA|genotype:UAS replicate1 GSM8775319 GSM8775319: Well P1 04A; Danio rerio; OTHER GSM8775319 r1 GSM8775319 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-04A.R1.fastq.gz P1-04A.R2.fastq.gz P1-04A_S25_I1_001.fastq.gz fastq fastq fastq 74926548.0 814419.0 GSM8775319 r1 0:34 1:48 2:10 A:14546606;C:5316463;G:9739043;T:9434376;N:55624 34 48 10   14546606 5316463 9739043 9434376 55624 SRX27572994 SRS23984647 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34743 34743 SRR32232955 SRX27572993 SRS23984646 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 11A GSM8775374   source name:6 dpf head|tissue:6 dpf head|well:P1 A11|rtindex:AATTAAGACT|genotype:GAL4UAS replicate3|geo loc name:missing|collection date:missing Well P1 11A Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 A11|rtindex:AATTAAGACT|genotype:GAL4UAS replicate3 GSM8775374 GSM8775374: Well P1 11A; Danio rerio; OTHER GSM8775374 r1 GSM8775374 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-11A.R1.fastq.gz P1-11A.R2.fastq.gz P1-11A_S81_I1_001.fastq.gz fastq fastq fastq 23002760.0 250030.0 GSM8775374 r1 0:34 1:48 2:10 A:3455540;C:1563605;G:4264428;T:2701802;N:16065 34 48 10   3455540 1563605 4264428 2701802 16065 SRX27572993 SRS23984646 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34744 34744 SRR32232956 SRX27572992 SRS23984645 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 10G GSM8775373   source name:6 dpf head|tissue:6 dpf head|well:P1 G10|rtindex:ATCCATGACT|genotype:wt replicate3|geo loc name:missing|collection date:missing Well P1 10G Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 G10|rtindex:ATCCATGACT|genotype:wt replicate3 GSM8775373 GSM8775373: Well P1 10G; Danio rerio; OTHER GSM8775373 r1 GSM8775373 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-10G.R1.fastq.gz P1-10G.R2.fastq.gz P1-10G_S79_I1_001.fastq.gz fastq fastq fastq 7274440.0 79070.0 GSM8775373 r1 0:34 1:48 2:10 A:1025995;C:453768;G:1424925;T:885556;N:5116 34 48 10   1025995 453768 1424925 885556 5116 SRX27572992 SRS23984645 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34745 34745 SRR32232957 SRX27572991 SRS23984644 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 10F GSM8775372   source name:6 dpf head|tissue:6 dpf head|well:P1 F10|rtindex:GCTGGAACTT|genotype:wt replicate3|geo loc name:missing|collection date:missing Well P1 10F Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 F10|rtindex:GCTGGAACTT|genotype:wt replicate3 GSM8775372 GSM8775372: Well P1 10F; Danio rerio; OTHER GSM8775372 r1 GSM8775372 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-10F.R1.fastq.gz P1-10F.R2.fastq.gz P1-10F_S78_I1_001.fastq.gz fastq fastq fastq 32953020.0 358185.0 GSM8775372 r1 0:34 1:48 2:10 A:5148026;C:2283765;G:5798660;T:3938086;N:24343 34 48 10   5148026 2283765 5798660 3938086 24343 SRX27572991 SRS23984644 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34746 34746 SRR32232958 SRX27572990 SRS23984643 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 10E GSM8775371   source name:6 dpf head|tissue:6 dpf head|well:P1 E10|rtindex:TATATACTAA|genotype:wt replicate3|geo loc name:missing|collection date:missing Well P1 10E Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 E10|rtindex:TATATACTAA|genotype:wt replicate3 GSM8775371 GSM8775371: Well P1 10E; Danio rerio; OTHER GSM8775371 r1 GSM8775371 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-10E.R1.fastq.gz P1-10E.R2.fastq.gz P1-10E_S77_I1_001.fastq.gz fastq fastq fastq 7366056084.0 80065827.0 GSM8775371 r1 0:34 1:48 2:10 A:1137867832;C:775309617;G:881567529;T:1042828519;N:5586199 34 48 10   1137867832 775309617 881567529 1042828519 5586199 SRX27572990 SRS23984643 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34747 34747 SRR32232959 SRX27572989 SRS23984642 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 10D GSM8775370   source name:6 dpf head|tissue:6 dpf head|well:P1 D10|rtindex:ACCGCCAACC|genotype:wt replicate3|geo loc name:missing|collection date:missing Well P1 10D Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 D10|rtindex:ACCGCCAACC|genotype:wt replicate3 GSM8775370 GSM8775370: Well P1 10D; Danio rerio; OTHER GSM8775370 r1 GSM8775370 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-10D.R1.fastq.gz P1-10D.R2.fastq.gz P1-10D_S76_I1_001.fastq.gz fastq fastq fastq 32482532.0 353071.0 GSM8775370 r1 0:34 1:48 2:10 A:4191254;C:2035744;G:6991172;T:3707782;N:21456 34 48 10   4191254 2035744 6991172 3707782 21456 SRX27572989 SRS23984642 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34748 34748 SRR32232960 SRX27572988 SRS23984641 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 10C GSM8775369   source name:6 dpf head|tissue:6 dpf head|well:P1 C10|rtindex:ACTTAACCTT|genotype:wt replicate3|geo loc name:missing|collection date:missing Well P1 10C Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 C10|rtindex:ACTTAACCTT|genotype:wt replicate3 GSM8775369 GSM8775369: Well P1 10C; Danio rerio; OTHER GSM8775369 r1 GSM8775369 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-10C.R1.fastq.gz P1-10C.R2.fastq.gz P1-10C_S75_I1_001.fastq.gz fastq fastq fastq 29742312.0 323286.0 GSM8775369 r1 0:34 1:48 2:10 A:4641549;C:2072399;G:5265185;T:3517381;N:21214 34 48 10   4641549 2072399 5265185 3517381 21214 SRX27572988 SRS23984641 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34749 34749 SRR32232961 SRX27572987 SRS23984640 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 10B GSM8775368   source name:6 dpf head|tissue:6 dpf head|well:P1 B10|rtindex:TTACCGAGGC|genotype:wt replicate3|geo loc name:missing|collection date:missing Well P1 10B Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 B10|rtindex:TTACCGAGGC|genotype:wt replicate3 GSM8775368 GSM8775368: Well P1 10B; Danio rerio; OTHER GSM8775368 r1 GSM8775368 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-10B.R1.fastq.gz P1-10B.R2.fastq.gz P1-10B_S74_I1_001.fastq.gz fastq fastq fastq 23657432.0 257146.0 GSM8775368 r1 0:34 1:48 2:10 A:3666551;C:1613324;G:4291430;T:2755799;N:15904 34 48 10   3666551 1613324 4291430 2755799 15904 SRX27572987 SRS23984640 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34750 34750 SRR32232962 SRX27572986 SRS23984639 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 10A GSM8775367   source name:6 dpf head|tissue:6 dpf head|well:P1 A10|rtindex:TAGATCTACT|genotype:wt replicate3|geo loc name:missing|collection date:missing Well P1 10A Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 A10|rtindex:TAGATCTACT|genotype:wt replicate3 GSM8775367 GSM8775367: Well P1 10A; Danio rerio; OTHER GSM8775367 r1 GSM8775367 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-10A.R1.fastq.gz P1-10A.R2.fastq.gz P1-10A_S73_I1_001.fastq.gz fastq fastq fastq 20690064.0 224892.0 GSM8775367 r1 0:34 1:48 2:10 A:3162846;C:1394801;G:3805546;T:2416575;N:15048 34 48 10   3162846 1394801 3805546 2416575 15048 SRX27572986 SRS23984639 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34751 34751 SRR32232963 SRX27572985 SRS23984638 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 06H GSM8775342   source name:6 dpf head|tissue:6 dpf head|well:P1 H06|rtindex:AGAGCCAAGG|genotype:wt replicate2|geo loc name:missing|collection date:missing Well P1 06H Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 H06|rtindex:AGAGCCAAGG|genotype:wt replicate2 GSM8775342 GSM8775342: Well P1 06H; Danio rerio; OTHER GSM8775342 r1 GSM8775342 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-06H.R1.fastq.gz P1-06H.R2.fastq.gz P1-06H_S48_I1_001.fastq.gz fastq fastq fastq 63664.0 692.0 GSM8775342 r1 0:34 1:48 2:10 A:13330;C:4382;G:7530;T:7889;N:85 34 48 10   13330 4382 7530 7889 85 SRX27572985 SRS23984638 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34752 34752 SRR32232964 SRX27572984 SRS23984637 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 06G GSM8775341   source name:6 dpf head|tissue:6 dpf head|well:P1 G06|rtindex:GGAGGATAGC|genotype:wt replicate2|geo loc name:missing|collection date:missing Well P1 06G Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 G06|rtindex:GGAGGATAGC|genotype:wt replicate2 GSM8775341 GSM8775341: Well P1 06G; Danio rerio; OTHER GSM8775341 r1 GSM8775341 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-06G.R1.fastq.gz P1-06G.R2.fastq.gz P1-06G_S47_I1_001.fastq.gz fastq fastq fastq 35465816.0 385498.0 GSM8775341 r1 0:34 1:48 2:10 A:5266761;C:2313383;G:6547005;T:4353297;N:23458 34 48 10   5266761 2313383 6547005 4353297 23458 SRX27572984 SRS23984637 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34753 34753 SRR32232965 SRX27572983 SRS23984636 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 06F GSM8775340   source name:6 dpf head|tissue:6 dpf head|well:P1 F06|rtindex:TCAACGTAAG|genotype:wt replicate2|geo loc name:missing|collection date:missing Well P1 06F Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 F06|rtindex:TCAACGTAAG|genotype:wt replicate2 GSM8775340 GSM8775340: Well P1 06F; Danio rerio; OTHER GSM8775340 r1 GSM8775340 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-06F.R1.fastq.gz P1-06F.R2.fastq.gz P1-06F_S46_I1_001.fastq.gz fastq fastq fastq 40023036.0 435033.0 GSM8775340 r1 0:34 1:48 2:10 A:6489618;C:2824422;G:6833067;T:4706650;N:27827 34 48 10   6489618 2824422 6833067 4706650 27827 SRX27572983 SRS23984636 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34754 34754 SRR32232966 SRX27572982 SRS23984635 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 06E GSM8775339   source name:6 dpf head|tissue:6 dpf head|well:P1 E06|rtindex:ACGCGAGATT|genotype:wt replicate2|geo loc name:missing|collection date:missing Well P1 06E Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 E06|rtindex:ACGCGAGATT|genotype:wt replicate2 GSM8775339 GSM8775339: Well P1 06E; Danio rerio; OTHER GSM8775339 r1 GSM8775339 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-06E.R1.fastq.gz P1-06E.R2.fastq.gz P1-06E_S45_I1_001.fastq.gz fastq fastq fastq 59610020.0 647935.0 GSM8775339 r1 0:34 1:48 2:10 A:10804415;C:4165808;G:9006405;T:7083045;N:41207 34 48 10   10804415 4165808 9006405 7083045 41207 SRX27572982 SRS23984635 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34755 34755 SRR32232967 SRX27572981 SRS23984633 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 06D GSM8775338   source name:6 dpf head|tissue:6 dpf head|well:P1 D06|rtindex:CATACTCCGA|genotype:wt replicate2|geo loc name:missing|collection date:missing Well P1 06D Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 D06|rtindex:CATACTCCGA|genotype:wt replicate2 GSM8775338 GSM8775338: Well P1 06D; Danio rerio; OTHER GSM8775338 r1 GSM8775338 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-06D.R1.fastq.gz P1-06D.R2.fastq.gz P1-06D_S44_I1_001.fastq.gz fastq fastq fastq 4944350388.0 53742939.0 GSM8775338 r1 0:34 1:48 2:10 A:924216784;C:417859577;G:514301359;T:719732920;N:3550432 34 48 10   924216784 417859577 514301359 719732920 3550432 SRX27572981 SRS23984633 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34756 34756 SRR32232968 SRX27572980 SRS23984634 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 06C GSM8775337   source name:6 dpf head|tissue:6 dpf head|well:P1 C06|rtindex:GCAGGTCCGT|genotype:wt replicate2|geo loc name:missing|collection date:missing Well P1 06C Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 C06|rtindex:GCAGGTCCGT|genotype:wt replicate2 GSM8775337 GSM8775337: Well P1 06C; Danio rerio; OTHER GSM8775337 r1 GSM8775337 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-06C.R1.fastq.gz P1-06C.R2.fastq.gz P1-06C_S43_I1_001.fastq.gz fastq fastq fastq 37857448.0 411494.0 GSM8775337 r1 0:34 1:48 2:10 A:6636308;C:2636497;G:5984141;T:4469989;N:24777 34 48 10   6636308 2636497 5984141 4469989 24777 SRX27572980 SRS23984634 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34757 34757 SRR32232969 SRX27572979 SRS23984632 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 06B GSM8775336   source name:6 dpf head|tissue:6 dpf head|well:P1 B06|rtindex:GGTACTGCCT|genotype:wt replicate2|geo loc name:missing|collection date:missing Well P1 06B Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 B06|rtindex:GGTACTGCCT|genotype:wt replicate2 GSM8775336 GSM8775336: Well P1 06B; Danio rerio; OTHER GSM8775336 r1 GSM8775336 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-06B.R1.fastq.gz P1-06B.R2.fastq.gz P1-06B_S42_I1_001.fastq.gz fastq fastq fastq 36989796.0 402063.0 GSM8775336 r1 0:34 1:48 2:10 A:6103502;C:2581732;G:6111203;T:4475906;N:26681 34 48 10   6103502 2581732 6111203 4475906 26681 SRX27572979 SRS23984632 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34758 34758 SRR32232970 SRX27572978 SRS23984631 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 06A GSM8775335   source name:6 dpf head|tissue:6 dpf head|well:P1 A06|rtindex:GCCGCAACGA|genotype:wt replicate2|geo loc name:missing|collection date:missing Well P1 06A Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 A06|rtindex:GCCGCAACGA|genotype:wt replicate2 GSM8775335 GSM8775335: Well P1 06A; Danio rerio; OTHER GSM8775335 r1 GSM8775335 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-06A.R1.fastq.gz P1-06A.R2.fastq.gz P1-06A_S41_I1_001.fastq.gz fastq fastq fastq 181733028.0 1975359.0 GSM8775335 r1 0:34 1:48 2:10 A:39079683;C:12211237;G:19042922;T:24349968;N:133422 34 48 10   39079683 12211237 19042922 24349968 133422 SRX27572978 SRS23984631 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34759 34759 SRR32232971 SRX27572977 SRS23984630 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 02H GSM8775310   source name:6 dpf head|tissue:6 dpf head|well:P1 H02|rtindex:TCTTGCCGAC|genotype:wt replicate1|geo loc name:missing|collection date:missing Well P1 02H Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 H02|rtindex:TCTTGCCGAC|genotype:wt replicate1 GSM8775310 GSM8775310: Well P1 02H; Danio rerio; OTHER GSM8775310 r1 GSM8775310 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-02H.R1.fastq.gz P1-02H.R2.fastq.gz P1-02H_S16_I1_001.fastq.gz fastq fastq fastq 460.0 5.0 GSM8775310 r1 0:34 1:48 2:10 A:79;C:22;G:97;T:42;N:0 34 48 10   79 22 97 42 0 SRX27572977 SRS23984630 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34760 34760 SRR32232972 SRX27572976 SRS23984629 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 02G GSM8775309   source name:6 dpf head|tissue:6 dpf head|well:P1 G02|rtindex:CGGATTAGAA|genotype:wt replicate1|geo loc name:missing|collection date:missing Well P1 02G Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 G02|rtindex:CGGATTAGAA|genotype:wt replicate1 GSM8775309 GSM8775309: Well P1 02G; Danio rerio; OTHER GSM8775309 r1 GSM8775309 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-02G.R1.fastq.gz P1-02G.R2.fastq.gz P1-02G_S15_I1_001.fastq.gz fastq fastq fastq 100724268.0 1094829.0 GSM8775309 r1 0:34 1:48 2:10 A:20865846;C:7071976;G:10244265;T:14295638;N:74067 34 48 10   20865846 7071976 10244265 14295638 74067 SRX27572976 SRS23984629 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34761 34761 SRR32232973 SRX27572975 SRS23984628 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 02F GSM8775308   source name:6 dpf head|tissue:6 dpf head|well:P1 F02|rtindex:CGTAGTTACC|genotype:wt replicate1|geo loc name:missing|collection date:missing Well P1 02F Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 F02|rtindex:CGTAGTTACC|genotype:wt replicate1 GSM8775308 GSM8775308: Well P1 02F; Danio rerio; OTHER GSM8775308 r1 GSM8775308 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-02F.R1.fastq.gz P1-02F.R2.fastq.gz P1-02F_S14_I1_001.fastq.gz fastq fastq fastq 397078348.0 4316069.0 GSM8775308 r1 0:34 1:48 2:10 A:92040782;C:26097382;G:36181559;T:52564018;N:287571 34 48 10   92040782 26097382 36181559 52564018 287571 SRX27572975 SRS23984628 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34762 34762 SRR32232974 SRX27572974 SRS23984627 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 02E GSM8775307   source name:6 dpf head|tissue:6 dpf head|well:P1 E02|rtindex:AACCTGCGTA|genotype:wt replicate1|geo loc name:missing|collection date:missing Well P1 02E Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 E02|rtindex:AACCTGCGTA|genotype:wt replicate1 GSM8775307 GSM8775307: Well P1 02E; Danio rerio; OTHER GSM8775307 r1 GSM8775307 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-02E.R1.fastq.gz P1-02E.R2.fastq.gz P1-02E_S13_I1_001.fastq.gz fastq fastq fastq 97598568.0 1060854.0 GSM8775307 r1 0:34 1:48 2:10 A:20986490;C:6417842;G:11267196;T:12179365;N:70099 34 48 10   20986490 6417842 11267196 12179365 70099 SRX27572974 SRS23984627 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34763 34763 SRR32232975 SRX27572973 SRS23984625 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 02D GSM8775306   source name:6 dpf head|tissue:6 dpf head|well:P1 D02|rtindex:GAAGATCGAG|genotype:wt replicate1|geo loc name:missing|collection date:missing Well P1 02D Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 D02|rtindex:GAAGATCGAG|genotype:wt replicate1 GSM8775306 GSM8775306: Well P1 02D; Danio rerio; OTHER GSM8775306 r1 GSM8775306 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-02D.R1.fastq.gz P1-02D.R2.fastq.gz P1-02D_S12_I1_001.fastq.gz fastq fastq fastq 4315783480.0 46910690.0 GSM8775306 r1 0:34 1:48 2:10 A:851820437;C:342635980;G:430524180;T:623563815;N:3168708 34 48 10   851820437 342635980 430524180 623563815 3168708 SRX27572973 SRS23984625 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34764 34764 SRR32232976 SRX27572972 SRS23984626 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 02C GSM8775305   source name:6 dpf head|tissue:6 dpf head|well:P1 C02|rtindex:CGACGCGACT|genotype:wt replicate1|geo loc name:missing|collection date:missing Well P1 02C Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 C02|rtindex:CGACGCGACT|genotype:wt replicate1 GSM8775305 GSM8775305: Well P1 02C; Danio rerio; OTHER GSM8775305 r1 GSM8775305 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-02C.R1.fastq.gz P1-02C.R2.fastq.gz P1-02C_S11_I1_001.fastq.gz fastq fastq fastq 62879608.0 683474.0 GSM8775305 r1 0:34 1:48 2:10 A:12210765;C:4319771;G:8466498;T:7766050;N:43668 34 48 10   12210765 4319771 8466498 7766050 43668 SRX27572972 SRS23984626 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34765 34765 SRR32232977 SRX27572971 SRS23984623 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 02B GSM8775304   source name:6 dpf head|tissue:6 dpf head|well:P1 B02|rtindex:AGAGCTATAA|genotype:wt replicate1|geo loc name:missing|collection date:missing Well P1 02B Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 B02|rtindex:AGAGCTATAA|genotype:wt replicate1 GSM8775304 GSM8775304: Well P1 02B; Danio rerio; OTHER GSM8775304 r1 GSM8775304 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-02B.R1.fastq.gz P1-02B.R2.fastq.gz P1-02B_S10_I1_001.fastq.gz fastq fastq fastq 43401920.0 471760.0 GSM8775304 r1 0:34 1:48 2:10 A:8440271;C:2935979;G:6044455;T:5195314;N:28461 34 48 10   8440271 2935979 6044455 5195314 28461 SRX27572971 SRS23984623 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34766 34766 SRR32232978 SRX27572970 SRS23984622 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 02A GSM8775303   source name:6 dpf head|tissue:6 dpf head|well:P1 A02|rtindex:GCGTTGGAGC|genotype:wt replicate1|geo loc name:missing|collection date:missing Well P1 02A Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 A02|rtindex:GCGTTGGAGC|genotype:wt replicate1 GSM8775303 GSM8775303: Well P1 02A; Danio rerio; OTHER GSM8775303 r1 GSM8775303 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-02A.R1.fastq.gz P1-02A.R2.fastq.gz P1-02A_S9_I1_001.fastq.gz fastq fastq fastq 461011540.0 5010995.0 GSM8775303 r1 0:34 1:48 2:10 A:104776330;C:30925787;G:42506931;T:61984548;N:334164 34 48 10   104776330 30925787 42506931 61984548 334164 SRX27572970 SRS23984622 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34767 34767 SRR32232979 SRX27572969 SRS23984624 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 12A GSM8775382   source name:6 dpf head|tissue:6 dpf head|well:P1 A12|rtindex:TTATTCATTC|genotype:UAS replicate3|geo loc name:missing|collection date:missing Well P1 12A Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 A12|rtindex:TTATTCATTC|genotype:UAS replicate3 GSM8775382 GSM8775382: Well P1 12A; Danio rerio; OTHER GSM8775382 r1 GSM8775382 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-12A.R1.fastq.gz P1-12A.R2.fastq.gz P1-12A_S89_I1_001.fastq.gz fastq fastq fastq 16741424.0 181972.0 GSM8775382 r1 0:34 1:48 2:10 A:2677910;C:1120864;G:2940806;T:1984541;N:10535 34 48 10   2677910 1120864 2940806 1984541 10535 SRX27572969 SRS23984624 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34768 34768 SRR32232980 SRX27572968 SRS23984621 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 11H GSM8775381   source name:6 dpf head|tissue:6 dpf head|well:P1 H11|rtindex:ACCTGGCCAA|genotype:GAL4UAS replicate3|geo loc name:missing|collection date:missing Well P1 11H Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 H11|rtindex:ACCTGGCCAA|genotype:GAL4UAS replicate3 GSM8775381 GSM8775381: Well P1 11H; Danio rerio; OTHER GSM8775381 r1 GSM8775381 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-11H.R1.fastq.gz P1-11H.R2.fastq.gz P1-11H_S88_I1_001.fastq.gz fastq fastq fastq 920.0 10.0 GSM8775381 r1 0:34 1:48 2:10 A:198;C:61;G:86;T:135;N:0 34 48 10   198 61 86 135 0 SRX27572968 SRS23984621 SRA2070568 UMass Chan UMass Chan                       T   under 1.2% mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34769 34769 SRR32232981 SRX27572967 SRS23984620 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 11G GSM8775380   source name:6 dpf head|tissue:6 dpf head|well:P1 G11|rtindex:AACCTGGTCT|genotype:GAL4UAS replicate3|geo loc name:missing|collection date:missing Well P1 11G Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 G11|rtindex:AACCTGGTCT|genotype:GAL4UAS replicate3 GSM8775380 GSM8775380: Well P1 11G; Danio rerio; OTHER GSM8775380 r1 GSM8775380 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-11G.R1.fastq.gz P1-11G.R2.fastq.gz P1-11G_S87_I1_001.fastq.gz fastq fastq fastq 47417168.0 515404.0 GSM8775380 r1 0:34 1:48 2:10 A:6801318;C:3146390;G:8860580;T:5896979;N:34125 34 48 10   6801318 3146390 8860580 5896979 34125 SRX27572967 SRS23984620 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34770 34770 SRR32232982 SRX27572966 SRS23984619 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 11F GSM8775379   source name:6 dpf head|tissue:6 dpf head|well:P1 F11|rtindex:AGGTTAGTTC|genotype:GAL4UAS replicate3|geo loc name:missing|collection date:missing Well P1 11F Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 F11|rtindex:AGGTTAGTTC|genotype:GAL4UAS replicate3 GSM8775379 GSM8775379: Well P1 11F; Danio rerio; OTHER GSM8775379 r1 GSM8775379 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-11F.R1.fastq.gz P1-11F.R2.fastq.gz P1-11F_S86_I1_001.fastq.gz fastq fastq fastq 31114768.0 338204.0 GSM8775379 r1 0:34 1:48 2:10 A:4790024;C:2139061;G:5605750;T:3678276;N:20681 34 48 10   4790024 2139061 5605750 3678276 20681 SRX27572966 SRS23984619 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34771 34771 SRR32232983 SRX27572965 SRS23984618 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 11E GSM8775378   source name:6 dpf head|tissue:6 dpf head|well:P1 E11|rtindex:ACTTGCTAGA|genotype:GAL4UAS replicate3|geo loc name:missing|collection date:missing Well P1 11E Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 E11|rtindex:ACTTGCTAGA|genotype:GAL4UAS replicate3 GSM8775378 GSM8775378: Well P1 11E; Danio rerio; OTHER GSM8775378 r1 GSM8775378 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-11E.R1.fastq.gz P1-11E.R2.fastq.gz P1-11E_S85_I1_001.fastq.gz fastq fastq fastq 30435440.0 330820.0 GSM8775378 r1 0:34 1:48 2:10 A:4748511;C:2122090;G:5400332;T:3588118;N:20309 34 48 10   4748511 2122090 5400332 3588118 20309 SRX27572965 SRS23984618 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34772 34772 SRR32232984 SRX27572964 SRS23984617 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 11D GSM8775377   source name:6 dpf head|tissue:6 dpf head|well:P1 D11|rtindex:ATAAGGAGCA|genotype:GAL4UAS replicate3|geo loc name:missing|collection date:missing Well P1 11D Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 D11|rtindex:ATAAGGAGCA|genotype:GAL4UAS replicate3 GSM8775377 GSM8775377: Well P1 11D; Danio rerio; OTHER GSM8775377 r1 GSM8775377 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-11D.R1.fastq.gz P1-11D.R2.fastq.gz P1-11D_S84_I1_001.fastq.gz fastq fastq fastq 28003972.0 304391.0 GSM8775377 r1 0:34 1:48 2:10 A:4493127;C:1907680;G:4963314;T:3228507;N:18140 34 48 10   4493127 1907680 4963314 3228507 18140 SRX27572964 SRS23984617 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34773 34773 SRR32232985 SRX27572963 SRS23984616 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 11C GSM8775376   source name:6 dpf head|tissue:6 dpf head|well:P1 C11|rtindex:CAACCGCTAA|genotype:GAL4UAS replicate3|geo loc name:missing|collection date:missing Well P1 11C Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 C11|rtindex:CAACCGCTAA|genotype:GAL4UAS replicate3 GSM8775376 GSM8775376: Well P1 11C; Danio rerio; OTHER GSM8775376 r1 GSM8775376 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-11C.R1.fastq.gz P1-11C.R2.fastq.gz P1-11C_S83_I1_001.fastq.gz fastq fastq fastq 21477952.0 233456.0 GSM8775376 r1 0:34 1:48 2:10 A:3270849;C:1474502;G:3915416;T:2530065;N:15056 34 48 10   3270849 1474502 3915416 2530065 15056 SRX27572963 SRS23984616 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34774 34774 SRR32232986 SRX27572962 SRS23984615 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 11B GSM8775375   source name:6 dpf head|tissue:6 dpf head|well:P1 B11|rtindex:ATCATATTAG|genotype:GAL4UAS replicate3|geo loc name:missing|collection date:missing Well P1 11B Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 B11|rtindex:ATCATATTAG|genotype:GAL4UAS replicate3 GSM8775375 GSM8775375: Well P1 11B; Danio rerio; OTHER GSM8775375 r1 GSM8775375 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-11B.R1.fastq.gz P1-11B.R2.fastq.gz P1-11B_S82_I1_001.fastq.gz fastq fastq fastq 21279600.0 231300.0 GSM8775375 r1 0:34 1:48 2:10 A:3177194;C:1463431;G:3934752;T:2512075;N:14948 34 48 10   3177194 1463431 3934752 2512075 14948 SRX27572962 SRS23984615 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34775 34775 SRR32232987 SRX27572961 SRS23984614 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 07H GSM8775350   source name:6 dpf head|tissue:6 dpf head|well:P1 H07|rtindex:AATACCATCC|genotype:GAL4UAS replicate2|geo loc name:missing|collection date:missing Well P1 07H Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 H07|rtindex:AATACCATCC|genotype:GAL4UAS replicate2 GSM8775350 GSM8775350: Well P1 07H; Danio rerio; OTHER GSM8775350 r1 GSM8775350 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-07H.R1.fastq.gz P1-07H.R2.fastq.gz P1-07H_S56_I1_001.fastq.gz fastq fastq fastq 644.0 7.0 GSM8775350 r1 0:34 1:48 2:10 A:110;C:48;G:94;T:84;N:0 34 48 10   110 48 94 84 0 SRX27572961 SRS23984614 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34776 34776 SRR32232988 SRX27572960 SRS23984612 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 07G GSM8775349   source name:6 dpf head|tissue:6 dpf head|well:P1 G07|rtindex:GGCTCTCTAT|genotype:GAL4UAS replicate2|geo loc name:missing|collection date:missing Well P1 07G Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 G07|rtindex:GGCTCTCTAT|genotype:GAL4UAS replicate2 GSM8775349 GSM8775349: Well P1 07G; Danio rerio; OTHER GSM8775349 r1 GSM8775349 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-07G.R1.fastq.gz P1-07G.R2.fastq.gz P1-07G_S55_I1_001.fastq.gz fastq fastq fastq 837346556.0 9101593.0 GSM8775349 r1 0:34 1:48 2:10 A:181851041;C:59310804;G:74090148;T:121010089;N:614382 34 48 10   181851041 59310804 74090148 121010089 614382 SRX27572960 SRS23984612 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34777 34777 SRR32232989 SRX27572959 SRS23984613 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 07F GSM8775348   source name:6 dpf head|tissue:6 dpf head|well:P1 F07|rtindex:TCTAATAGTA|genotype:GAL4UAS replicate2|geo loc name:missing|collection date:missing Well P1 07F Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 F07|rtindex:TCTAATAGTA|genotype:GAL4UAS replicate2 GSM8775348 GSM8775348: Well P1 07F; Danio rerio; OTHER GSM8775348 r1 GSM8775348 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-07F.R1.fastq.gz P1-07F.R2.fastq.gz P1-07F_S54_I1_001.fastq.gz fastq fastq fastq 51998308.0 565199.0 GSM8775348 r1 0:34 1:48 2:10 A:8691899;C:3542473;G:8590067;T:6268755;N:36358 34 48 10   8691899 3542473 8590067 6268755 36358 SRX27572959 SRS23984613 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34778 34778 SRR32232990 SRX27572958 SRS23984611 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 07E GSM8775347   source name:6 dpf head|tissue:6 dpf head|well:P1 E07|rtindex:GGTATCCGCC|genotype:GAL4UAS replicate2|geo loc name:missing|collection date:missing Well P1 07E Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 E07|rtindex:GGTATCCGCC|genotype:GAL4UAS replicate2 GSM8775347 GSM8775347: Well P1 07E; Danio rerio; OTHER GSM8775347 r1 GSM8775347 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-07E.R1.fastq.gz P1-07E.R2.fastq.gz P1-07E_S53_I1_001.fastq.gz fastq fastq fastq 4757958664.0 51716942.0 GSM8775347 r1 0:34 1:48 2:10 A:917767286;C:388310145;G:453981093;T:718871529;N:3483163 34 48 10   917767286 388310145 453981093 718871529 3483163 SRX27572958 SRS23984611 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34779 34779 SRR32232991 SRX27572957 SRS23984610 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 07D GSM8775346   source name:6 dpf head|tissue:6 dpf head|well:P1 D07|rtindex:GCTAACTTGC|genotype:GAL4UAS replicate2|geo loc name:missing|collection date:missing Well P1 07D Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 D07|rtindex:GCTAACTTGC|genotype:GAL4UAS replicate2 GSM8775346 GSM8775346: Well P1 07D; Danio rerio; OTHER GSM8775346 r1 GSM8775346 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-07D.R1.fastq.gz P1-07D.R2.fastq.gz P1-07D_S52_I1_001.fastq.gz fastq fastq fastq 33692056.0 366218.0 GSM8775346 r1 0:34 1:48 2:10 A:5438617;C:2339082;G:5799183;T:3978938;N:22644 34 48 10   5438617 2339082 5799183 3978938 22644 SRX27572957 SRS23984610 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34780 34780 SRR32232992 SRX27572956 SRS23984609 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 07C GSM8775345   source name:6 dpf head|tissue:6 dpf head|well:P1 C07|rtindex:TCGGCCTTAC|genotype:GAL4UAS replicate2|geo loc name:missing|collection date:missing Well P1 07C Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 C07|rtindex:TCGGCCTTAC|genotype:GAL4UAS replicate2 GSM8775345 GSM8775345: Well P1 07C; Danio rerio; OTHER GSM8775345 r1 GSM8775345 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-07C.R1.fastq.gz P1-07C.R2.fastq.gz P1-07C_S51_I1_001.fastq.gz fastq fastq fastq 1594757348.0 17334319.0 GSM8775345 r1 0:34 1:48 2:10 A:266696003;C:139558591;G:158516635;T:266107758;N:1168325 34 48 10   266696003 139558591 158516635 266107758 1168325 SRX27572956 SRS23984609 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34781 34781 SRR32232993 SRX27572955 SRS23984608 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 07B GSM8775344   source name:6 dpf head|tissue:6 dpf head|well:P1 B07|rtindex:TAGAATTAAC|genotype:GAL4UAS replicate2|geo loc name:missing|collection date:missing Well P1 07B Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 B07|rtindex:TAGAATTAAC|genotype:GAL4UAS replicate2 GSM8775344 GSM8775344: Well P1 07B; Danio rerio; OTHER GSM8775344 r1 GSM8775344 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-07B.R1.fastq.gz P1-07B.R2.fastq.gz P1-07B_S50_I1_001.fastq.gz fastq fastq fastq 30341600.0 329800.0 GSM8775344 r1 0:34 1:48 2:10 A:4935051;C:2157330;G:5157156;T:3559714;N:21149 34 48 10   4935051 2157330 5157156 3559714 21149 SRX27572955 SRS23984608 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34782 34782 SRR32232994 SRX27572954 SRS23984607 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 07A GSM8775343   source name:6 dpf head|tissue:6 dpf head|well:P1 A07|rtindex:TGAGTCTGGC|genotype:GAL4UAS replicate2|geo loc name:missing|collection date:missing Well P1 07A Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 A07|rtindex:TGAGTCTGGC|genotype:GAL4UAS replicate2 GSM8775343 GSM8775343: Well P1 07A; Danio rerio; OTHER GSM8775343 r1 GSM8775343 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-07A.R1.fastq.gz P1-07A.R2.fastq.gz P1-07A_S49_I1_001.fastq.gz fastq fastq fastq 51962060.0 564805.0 GSM8775343 r1 0:34 1:48 2:10 A:9098744;C:3684168;G:8377271;T:5913518;N:36939 34 48 10   9098744 3684168 8377271 5913518 36939 SRX27572954 SRS23984607 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34783 34783 SRR32232995 SRX27572953 SRS23984606 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 03H GSM8775318   source name:6 dpf head|tissue:6 dpf head|well:P1 H03|rtindex:AAGACCGTTG|genotype:GAL4UAS replicate1|geo loc name:missing|collection date:missing Well P1 03H Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 H03|rtindex:AAGACCGTTG|genotype:GAL4UAS replicate1 GSM8775318 GSM8775318: Well P1 03H; Danio rerio; OTHER GSM8775318 r1 GSM8775318 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-03H.R1.fastq.gz P1-03H.R2.fastq.gz P1-03H_S24_I1_001.fastq.gz fastq fastq fastq 1012.0 11.0 GSM8775318 r1 0:34 1:48 2:10 A:194;C:60;G:140;T:134;N:0 34 48 10   194 60 140 134 0 SRX27572953 SRS23984606 SRA2070568 UMass Chan UMass Chan                             illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34784 34784 SRR32232996 SRX27572952 SRS23984605 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 03G GSM8775317   source name:6 dpf head|tissue:6 dpf head|well:P1 G03|rtindex:ATCGGCTATC|genotype:GAL4UAS replicate1|geo loc name:missing|collection date:missing Well P1 03G Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 G03|rtindex:ATCGGCTATC|genotype:GAL4UAS replicate1 GSM8775317 GSM8775317: Well P1 03G; Danio rerio; OTHER GSM8775317 r1 GSM8775317 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-03G.R1.fastq.gz P1-03G.R2.fastq.gz P1-03G_S23_I1_001.fastq.gz fastq fastq fastq 10973024.0 119272.0 GSM8775317 r1 0:34 1:48 2:10 A:1618652;C:710901;G:2106027;T:1282180;N:7296 34 48 10   1618652 710901 2106027 1282180 7296 SRX27572952 SRS23984605 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34785 34785 SRR32232997 SRX27572951 SRS23984604 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 03F GSM8775316   source name:6 dpf head|tissue:6 dpf head|well:P1 F03|rtindex:TCCAATCATC|genotype:GAL4UAS replicate1|geo loc name:missing|collection date:missing Well P1 03F Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 F03|rtindex:TCCAATCATC|genotype:GAL4UAS replicate1 GSM8775316 GSM8775316: Well P1 03F; Danio rerio; OTHER GSM8775316 r1 GSM8775316 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-03F.R1.fastq.gz P1-03F.R2.fastq.gz P1-03F_S22_I1_001.fastq.gz fastq fastq fastq 143677228.0 1561709.0 GSM8775316 r1 0:34 1:48 2:10 A:32106494;C:9327944;G:15133487;T:18290237;N:103870 34 48 10   32106494 9327944 15133487 18290237 103870 SRX27572951 SRS23984604 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34786 34786 SRR32232998 SRX27572950 SRS23984603 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 03E GSM8775315   source name:6 dpf head|tissue:6 dpf head|well:P1 E03|rtindex:GGCAGACGCC|genotype:GAL4UAS replicate1|geo loc name:missing|collection date:missing Well P1 03E Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 E03|rtindex:GGCAGACGCC|genotype:GAL4UAS replicate1 GSM8775315 GSM8775315: Well P1 03E; Danio rerio; OTHER GSM8775315 r1 GSM8775315 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-03E.R1.fastq.gz P1-03E.R2.fastq.gz P1-03E_S21_I1_001.fastq.gz fastq fastq fastq 140146084.0 1523327.0 GSM8775315 r1 0:34 1:48 2:10 A:31463127;C:9060717;G:14802782;T:17693205;N:99865 34 48 10   31463127 9060717 14802782 17693205 99865 SRX27572950 SRS23984603 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34787 34787 SRR32232999 SRX27572949 SRS23984602 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 03D GSM8775314   source name:6 dpf head|tissue:6 dpf head|well:P1 D03|rtindex:AAGAAGCTAG|genotype:GAL4UAS replicate1|geo loc name:missing|collection date:missing Well P1 03D Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 D03|rtindex:AAGAAGCTAG|genotype:GAL4UAS replicate1 GSM8775314 GSM8775314: Well P1 03D; Danio rerio; OTHER GSM8775314 r1 GSM8775314 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-03D.R1.fastq.gz P1-03D.R2.fastq.gz P1-03D_S20_I1_001.fastq.gz fastq fastq fastq 37007184.0 402252.0 GSM8775314 r1 0:34 1:48 2:10 A:6392785;C:2627202;G:5968712;T:4292408;N:26989 34 48 10   6392785 2627202 5968712 4292408 26989 SRX27572949 SRS23984602 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34788 34788 SRR32233000 SRX27572948 SRS23984601 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 03C GSM8775313   source name:6 dpf head|tissue:6 dpf head|well:P1 C03|rtindex:GATACGGAAC|genotype:GAL4UAS replicate1|geo loc name:missing|collection date:missing Well P1 03C Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 C03|rtindex:GATACGGAAC|genotype:GAL4UAS replicate1 GSM8775313 GSM8775313: Well P1 03C; Danio rerio; OTHER GSM8775313 r1 GSM8775313 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-03C.R1.fastq.gz P1-03C.R2.fastq.gz P1-03C_S19_I1_001.fastq.gz fastq fastq fastq 310907376.0 3379428.0 GSM8775313 r1 0:34 1:48 2:10 A:71568831;C:20485574;G:28605447;T:41329422;N:223270 34 48 10   71568831 20485574 28605447 41329422 223270 SRX27572948 SRS23984601 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34789 34789 SRR32233001 SRX27572947 SRS23984600 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 03B GSM8775312   source name:6 dpf head|tissue:6 dpf head|well:P1 B03|rtindex:CTAAGAGAAG|genotype:GAL4UAS replicate1|geo loc name:missing|collection date:missing Well P1 03B Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 B03|rtindex:CTAAGAGAAG|genotype:GAL4UAS replicate1 GSM8775312 GSM8775312: Well P1 03B; Danio rerio; OTHER GSM8775312 r1 GSM8775312 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-03B.R1.fastq.gz P1-03B.R2.fastq.gz P1-03B_S18_I1_001.fastq.gz fastq fastq fastq 45819128.0 498034.0 GSM8775312 r1 0:34 1:48 2:10 A:7950763;C:3191236;G:7350319;T:5380702;N:32612 34 48 10   7950763 3191236 7350319 5380702 32612 SRX27572947 SRS23984600 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34790 34790 SRR32233002 SRX27572946 SRS23984599 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 03A GSM8775311   source name:6 dpf head|tissue:6 dpf head|well:P1 A03|rtindex:GATCTTACGC|genotype:GAL4UAS replicate1|geo loc name:missing|collection date:missing Well P1 03A Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 A03|rtindex:GATCTTACGC|genotype:GAL4UAS replicate1 GSM8775311 GSM8775311: Well P1 03A; Danio rerio; OTHER GSM8775311 r1 GSM8775311 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-03A.R1.fastq.gz P1-03A.R2.fastq.gz P1-03A_S17_I1_001.fastq.gz fastq fastq fastq 637838760.0 6933030.0 GSM8775311 r1 0:34 1:48 2:10 A:143046137;C:43682277;G:61320584;T:84263744;N:472698 34 48 10   143046137 43682277 61320584 84263744 472698 SRX27572946 SRS23984599 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34791 34791 SRR32233003 SRX27572945 SRS23984598 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 01H GSM8775302   source name:6 dpf head|tissue:6 dpf head|well:P1 H01|rtindex:AAGCCAGTTA|genotype:GAL4 replicate1|geo loc name:missing|collection date:missing Well P1 01H Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 H01|rtindex:AAGCCAGTTA|genotype:GAL4 replicate1 GSM8775302 GSM8775302: Well P1 01H; Danio rerio; OTHER GSM8775302 r1 GSM8775302 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-01H.R1.fastq.gz P1-01H.R2.fastq.gz P1-01H_S8_I1_001.fastq.gz fastq fastq fastq 460.0 5.0 GSM8775302 r1 0:34 1:48 2:10 A:117;C:22;G:25;T:76;N:0 34 48 10   117 22 25 76 0 SRX27572945 SRS23984598 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous System
34792 34792 SRR32233004 SRX27572944 SRS23984597 SRP561592 PRJNA1219235 Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4 GSE288775 Transcriptome Analysis The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.       Well P1 01G GSM8775301   source name:6 dpf head|tissue:6 dpf head|well:P1 G01|rtindex:CATTCAATCA|genotype:GAL4 replicate1|geo loc name:missing|collection date:missing Well P1 01G Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq 6 dpf head   tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2. Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping. tissue:6 dpf head|well:P1 G01|rtindex:CATTCAATCA|genotype:GAL4 replicate1 GSM8775301 GSM8775301: Well P1 01G; Danio rerio; OTHER GSM8775301 r1 GSM8775301 1 tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.   OTHER TRANSCRIPTOMIC other PAIRED ILLUMINA NextSeq 500   SRP561592   loader:fastq load.py P1-01G.R1.fastq.gz P1-01G.R2.fastq.gz P1-01G_S7_I1_001.fastq.gz fastq fastq fastq 61910756.0 672943.0 GSM8775301 r1 0:34 1:48 2:10 A:11472187;C:4248935;G:8848756;T:7686532;N:44854 34 48 10   11472187 4248935 8848756 7686532 44854 SRX27572944 SRS23984597 SRA2070568 UMass Chan UMass Chan                       B   usable mapping rate illumina nextseq unknown other unknown sc single_cell_plate scirnaseq   United States 2025-02-04 Larval Larval Head Nervous 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");;