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
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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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,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
34793,SRR32233005,SRX27572943,SRS23984596,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 01F,GSM8775300,,source name:6 dpf head|tissue:6 dpf head|well:P1 F01|rtindex:TCGCGGTTGG|genotype:GAL4 replicate1|geo loc name:missing|collection date:missing,Well P1 01F,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 F01|rtindex:TCGCGGTTGG|genotype:GAL4 replicate1,GSM8775300,GSM8775300: Well P1 01F; Danio rerio; OTHER,GSM8775300 r1,GSM8775300,1,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,P1-01F.R1.fastq.gz P1-01F.R2.fastq.gz P1-01F_S6_I1_001.fastq.gz,fastq fastq fastq,47886460.0,520505.0,GSM8775300 r1,0:34 1:48 2:10,A:7538322;C:3307775;G:8680182;T:5423914;N:34047,34,48,10,,7538322,3307775,8680182,5423914,34047,SRX27572943,SRS23984596,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
34794,SRR32233006,SRX27572942,SRS23984595,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 01E,GSM8775299,,source name:6 dpf head|tissue:6 dpf head|well:P1 E01|rtindex:GGTATGCTTG|genotype:GAL4 replicate1|geo loc name:missing|collection date:missing,Well P1 01E,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 E01|rtindex:GGTATGCTTG|genotype:GAL4 replicate1,GSM8775299,GSM8775299: Well P1 01E; Danio rerio; OTHER,GSM8775299 r1,GSM8775299,1,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,P1-01E.R1.fastq.gz P1-01E.R2.fastq.gz P1-01E_S5_I1_001.fastq.gz,fastq fastq fastq,316428848.0,3439444.0,GSM8775299 r1,0:34 1:48 2:10,A:73583462;C:20434592;G:30034092;T:40809909;N:231257,34,48,10,,73583462,20434592,30034092,40809909,231257,SRX27572942,SRS23984595,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
34795,SRR32233007,SRX27572941,SRS23984594,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 01D,GSM8775298,,source name:6 dpf head|tissue:6 dpf head|well:P1 D01|rtindex:TCTGATACCA|genotype:GAL4 replicate1|geo loc name:missing|collection date:missing,Well P1 01D,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 D01|rtindex:TCTGATACCA|genotype:GAL4 replicate1,GSM8775298,GSM8775298: Well P1 01D; Danio rerio; OTHER,GSM8775298 r1,GSM8775298,1,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,P1-01D.R1.fastq.gz P1-01D.R2.fastq.gz P1-01D_S4_I1_001.fastq.gz,fastq fastq fastq,45210456.0,491418.0,GSM8775298 r1,0:34 1:48 2:10,A:6229214;C:2881227;G:9440659;T:5007385;N:29579,34,48,10,,6229214,2881227,9440659,5007385,29579,SRX27572941,SRS23984594,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
34796,SRR32233008,SRX27572940,SRS23984593,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 01C,GSM8775297,,source name:6 dpf head|tissue:6 dpf head|well:P1 C01|rtindex:ACTATGCAAT|genotype:GAL4 replicate1|geo loc name:missing|collection date:missing,Well P1 01C,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 C01|rtindex:ACTATGCAAT|genotype:GAL4 replicate1,GSM8775297,GSM8775297: Well P1 01C; Danio rerio; OTHER,GSM8775297 r1,GSM8775297,1,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,P1-01C.R1.fastq.gz P1-01C.R2.fastq.gz P1-01C_S3_I1_001.fastq.gz,fastq fastq fastq,1882930328.0,20466634.0,GSM8775297 r1,0:34 1:48 2:10,A:399637116;C:138660698;G:172248178;T:270484119;N:1368321,34,48,10,,399637116,138660698,172248178,270484119,1368321,SRX27572940,SRS23984593,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
34797,SRR32233009,SRX27572939,SRS23984592,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 01B,GSM8775296,,source name:6 dpf head|tissue:6 dpf head|well:P1 B01|rtindex:TTGACTTCAG|genotype:GAL4 replicate1|geo loc name:missing|collection date:missing,Well P1 01B,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 B01|rtindex:TTGACTTCAG|genotype:GAL4 replicate1,GSM8775296,GSM8775296: Well P1 01B; Danio rerio; OTHER,GSM8775296 r1,GSM8775296,1,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,P1-01B.R1.fastq.gz P1-01B.R2.fastq.gz P1-01B_S2_I1_001.fastq.gz,fastq fastq fastq,41624480.0,452440.0,GSM8775296 r1,0:34 1:48 2:10,A:7285464;C:2932880;G:6556513;T:4915495;N:26768,34,48,10,,7285464,2932880,6556513,4915495,26768,SRX27572939,SRS23984592,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
34798,SRR32233010,SRX27572938,SRS23984591,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 01A,GSM8775295,,source name:6 dpf head|tissue:6 dpf head|well:P1 A01|rtindex:TCCTACCAGT|genotype:GAL4 replicate1|geo loc name:missing|collection date:missing,Well P1 01A,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 A01|rtindex:TCCTACCAGT|genotype:GAL4 replicate1,GSM8775295,GSM8775295: Well P1 01A; Danio rerio; OTHER,GSM8775295 r1,GSM8775295,1,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,P1-01A.R1.fastq.gz P1-01A.R2.fastq.gz P1-01A_S1_I1_001.fastq.gz,fastq fastq fastq,42216040.0,458870.0,GSM8775295 r1,0:34 1:48 2:10,A:6917417;C:3128899;G:6813315;T:5136649;N:29480,34,48,10,,6917417,3128899,6813315,5136649,29480,SRX27572938,SRS23984591,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
34799,SRR32232916,SRX27572937,SRS23984589,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 09H,GSM8775366,,source name:6 dpf head|tissue:6 dpf head|well:P1 H09|rtindex:AGCTTATCCG|genotype:GAL4 replicate3|geo loc name:missing|collection date:missing,Well P1 09H,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 H09|rtindex:AGCTTATCCG|genotype:GAL4 replicate3,GSM8775366,GSM8775366: Well P1 09H; Danio rerio; OTHER,GSM8775366 r1,GSM8775366,1,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,P1-09H.R1.fastq.gz P1-09H.R2.fastq.gz P1-09H_S72_I1_001.fastq.gz,fastq fastq fastq,828.0,9.0,GSM8775366 r1,0:34 1:48 2:10,A:157;C:46;G:114;T:115;N:0,34,48,10,,157,46,114,115,0,SRX27572937,SRS23984589,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
34800,SRR32232917,SRX27572936,SRS23984590,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 09G,GSM8775365,,source name:6 dpf head|tissue:6 dpf head|well:P1 G09|rtindex:CGCTCCTAAC|genotype:GAL4 replicate3|geo loc name:missing|collection date:missing,Well P1 09G,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 G09|rtindex:CGCTCCTAAC|genotype:GAL4 replicate3,GSM8775365,GSM8775365: Well P1 09G; Danio rerio; OTHER,GSM8775365 r1,GSM8775365,1,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,P1-09G.R1.fastq.gz P1-09G.R2.fastq.gz P1-09G_S71_I1_001.fastq.gz,fastq fastq fastq,1572464.0,17092.0,GSM8775365 r1,0:34 1:48 2:10,A:211725;C:88504;G:340138;T:179085;N:964,34,48,10,,211725,88504,340138,179085,964,SRX27572936,SRS23984590,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
34801,SRR32232918,SRX27572935,SRS23984588,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 09F,GSM8775364,,source name:6 dpf head|tissue:6 dpf head|well:P1 F09|rtindex:CTAACTAGAT|genotype:GAL4 replicate3|geo loc name:missing|collection date:missing,Well P1 09F,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 F09|rtindex:CTAACTAGAT|genotype:GAL4 replicate3,GSM8775364,GSM8775364: Well P1 09F; Danio rerio; OTHER,GSM8775364 r1,GSM8775364,1,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,P1-09F.R1.fastq.gz P1-09F.R2.fastq.gz P1-09F_S70_I1_001.fastq.gz,fastq fastq fastq,39343984.0,427652.0,GSM8775364 r1,0:34 1:48 2:10,A:6502523;C:2717878;G:6622725;T:4655575;N:28595,34,48,10,,6502523,2717878,6622725,4655575,28595,SRX27572935,SRS23984588,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
34802,SRR32232919,SRX27572934,SRS23984587,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 09E,GSM8775363,,source name:6 dpf head|tissue:6 dpf head|well:P1 E09|rtindex:TCGCTAAGCA|genotype:GAL4 replicate3|geo loc name:missing|collection date:missing,Well P1 09E,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 E09|rtindex:TCGCTAAGCA|genotype:GAL4 replicate3,GSM8775363,GSM8775363: Well P1 09E; Danio rerio; OTHER,GSM8775363 r1,GSM8775363,1,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,P1-09E.R1.fastq.gz P1-09E.R2.fastq.gz P1-09E_S69_I1_001.fastq.gz,fastq fastq fastq,24184408.0,262874.0,GSM8775363 r1,0:34 1:48 2:10,A:3491757;C:1565663;G:4816947;T:2727737;N:15848,34,48,10,,3491757,1565663,4816947,2727737,15848,SRX27572934,SRS23984587,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
34803,SRR32232920,SRX27572933,SRS23984586,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 09D,GSM8775362,,source name:6 dpf head|tissue:6 dpf head|well:P1 D09|rtindex:CCGATTCCTG|genotype:GAL4 replicate3|geo loc name:missing|collection date:missing,Well P1 09D,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 D09|rtindex:CCGATTCCTG|genotype:GAL4 replicate3,GSM8775362,GSM8775362: Well P1 09D; Danio rerio; OTHER,GSM8775362 r1,GSM8775362,1,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,P1-09D.R1.fastq.gz P1-09D.R2.fastq.gz P1-09D_S68_I1_001.fastq.gz,fastq fastq fastq,8805688.0,95714.0,GSM8775362 r1,0:34 1:48 2:10,A:1193944;C:510582;G:1896290;T:987138;N:6318,34,48,10,,1193944,510582,1896290,987138,6318,SRX27572933,SRS23984586,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
34804,SRR32232921,SRX27572932,SRS23984585,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 09C,GSM8775361,,source name:6 dpf head|tissue:6 dpf head|well:P1 C09|rtindex:CCAGTTCCAA|genotype:GAL4 replicate3|geo loc name:missing|collection date:missing,Well P1 09C,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 C09|rtindex:CCAGTTCCAA|genotype:GAL4 replicate3,GSM8775361,GSM8775361: Well P1 09C; Danio rerio; OTHER,GSM8775361 r1,GSM8775361,1,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,P1-09C.R1.fastq.gz P1-09C.R2.fastq.gz P1-09C_S67_I1_001.fastq.gz,fastq fastq fastq,7000372.0,76091.0,GSM8775361 r1,0:34 1:48 2:10,A:930135;C:403236;G:1527971;T:786387;N:4639,34,48,10,,930135,403236,1527971,786387,4639,SRX27572932,SRS23984585,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
34805,SRR32232922,SRX27572931,SRS23984584,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 09B,GSM8775360,,source name:6 dpf head|tissue:6 dpf head|well:P1 B09|rtindex:TGCCGGCAGA|genotype:GAL4 replicate3|geo loc name:missing|collection date:missing,Well P1 09B,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 B09|rtindex:TGCCGGCAGA|genotype:GAL4 replicate3,GSM8775360,GSM8775360: Well P1 09B; Danio rerio; OTHER,GSM8775360 r1,GSM8775360,1,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,P1-09B.R1.fastq.gz P1-09B.R2.fastq.gz P1-09B_S66_I1_001.fastq.gz,fastq fastq fastq,9253268.0,100579.0,GSM8775360 r1,0:34 1:48 2:10,A:1246519;C:557682;G:1976082;T:1040959;N:6550,34,48,10,,1246519,557682,1976082,1040959,6550,SRX27572931,SRS23984584,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
34806,SRR32232923,SRX27572930,SRS23984583,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 09A,GSM8775359,,source name:6 dpf head|tissue:6 dpf head|well:P1 A09|rtindex:ACGGAGGCGG|genotype:GAL4 replicate3|geo loc name:missing|collection date:missing,Well P1 09A,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 A09|rtindex:ACGGAGGCGG|genotype:GAL4 replicate3,GSM8775359,GSM8775359: Well P1 09A; Danio rerio; OTHER,GSM8775359 r1,GSM8775359,1,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,P1-09A.R1.fastq.gz P1-09A.R2.fastq.gz P1-09A_S65_I1_001.fastq.gz,fastq fastq fastq,19339780.0,210215.0,GSM8775359 r1,0:34 1:48 2:10,A:2845395;C:1284021;G:3694044;T:2253744;N:13116,34,48,10,,2845395,1284021,3694044,2253744,13116,SRX27572930,SRS23984583,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
69693,SRR19155732,SRX15222308,SRS12956194,SRP374541,PRJNA836866,Embryo scale reverse genetics at single cell resolution,GSE202639,Transcriptome Analysis,The maturation of single cell transcriptomic technologies has facilitated the generation of comprehensive cellular atlases from whole embryos. A majority of this data  however  has been collected from wild type embryos without xxx appreciation for latent variation present in development. Here we present single cell transcriptomic data from 1812 individually resolved developing zebrafish embryos  encompassing 19 time points  23 genetic perturbations  and totaling 3.2M cells. The high degree of replication in our study 8 or more embryos per condition allows us to estimate the variance in cell type abundance organism wide and to detect perturbation dependent deviance in cell type composition relative to wild type embryos. Our approach is sensitive to rare cell types  resolving developmental trajectories and genetic dependencies in the cranial ganglia neurons  a cell population that comprises less than 1% of the embryo. Additionally  time series profiling of individual mutants identified a group of brachyury independent cells with strikingly similar transcriptomes to notochord sheath cells  leading to new hypotheses about the origins of the skull. We anticipate that standardized collection of high resolution  organism scale single cell data from large numbers of individual embryos will enable mapping the genetic dependencies of zebrafish cell types  while also addressing long standing challenges in developmental genetics  including the cellular and transcriptional plasticity underlying phenotypic diversity across individuals. Overall design: Whole zebrafish embryos were collected across multiple timepoints or in response to genetic perturbations with sci RNA seq3. Please note that the following processed data files have been updated on Mar 7  2023: zperturb full cell metadata.csv zperturb full gene metadata.csv zperturb full raw counts.RDS,,pubmed:37968389,,Zebrafish perturbation atlas plates4 6,GSM6127483,,tissue:Whole embryo|cell type:Whole embryo|developmental stage:18  24  36  48  72 hpf|strain:AB|genotype:ctrl cr  tbx16  tbx16 tbx16l  tbx16 msgn1  cdx4  cdx4 cdx1a  tbxta  tbx16  tbx16l  msgn1  wnt3a wnt8a  noto  smo  tbx1  hand2  egr2b  epha4a  hoxb1a  mafba  zc4h2  phox2a  foxi1  hgfa  met  foxd3  tfap2a  tfap2a foxd3,Zebrafish perturbation atlas plates4 6,The demultiplexing  barcoded processing  gene counting and aggregation were performed using the Brotman Baty sci RNA seq demultiplexing and processing pipelines https://github.com/bbi lab/bbi dmux; https://github.com/bbi lab/bbi sci. Count matrix was loaded into Monocle3 for further analysis Assembly: GRCz11 Supplementary files format and content: Count Matrix RDS file  Cell Metadata comma separated values  .csv  Gene Metadata comma separated values  .csv Supplementary files format and content: zperturb pilot cds.RDS monocle3 cell data set object Supplementary files format and content: zperturb pilot raw counts.RDS dgCMatrix with raw counts Supplementary files format and content: zperturb pilot cell metadata.csv cell metadata table comma separated Supplementary files format and content: zperturb pilot gene metadata.csv gene metadata table comma separated Supplementary files format and content: zperturb pilot hashTable.txt hash oligo count table Supplementary files format and content: reference cds.RDS monocle3 cell data set object Supplementary files format and content: reference raw counts.RDS dgCMatrix with raw counts Supplementary files format and content: reference cell metadata.csv cell metadata table comma separated Supplementary files format and content: reference gene metadata.csv gene metadata table comma separated Supplementary files format and content: reference expt1 hashTable.txt hash oligo count table Supplementary files format and content: reference expt2 hashTable.txt hash oligo count table Supplementary files format and content: reference expt3 hashTable.txt hash oligo count table Supplementary files format and content: zperturb full cds.RDS monocle3 cell data set object Supplementary files format and content: zperturb full raw counts.RDS dgCMatrix with raw counts Supplementary files format and content: zperturb full cell metadata.csv cell metadata table comma separated Supplementary files format and content: zperturb full gene metadata.csv gene metadata table comma separated Supplementary files format and content: zperturb full run1 hashTable.txt hash oligo count table Supplementary files format and content: zperturb full run2 hashTable.txt hash oligo count table,Whole embryo,,Embryos were transferred  one by one  into separate wells of a 96 well V bottom plate containing 75μL of 1X TrypLE + 2 mg/mL Collagenase P. Embryos were then dissociated by manual trituration at 30℃ once every 5 minutes. Dissociation continued until no visible chunks were present under a dissecting scope  which took between 20 40 minutes depending on embryo stage e.g. 20 minutes for 18hpf and 40 minutes for 72hpf. Stop solution [1x dPBS  5% FBS ] was then added to each well to quench the proteases. three prime end capture of polyAdenylated transcripts. In situ reverse transcription with barcoded RT primers followed by second strand synthesis  tagmentation and index PCR gives each nucleus and all the RNA molecules contained within it  a unique combination of barcode sequences. These barcodes are then demultiplexed to create single cells in silico.,Zebrafish embryos were raised in embryo media at 28.5C.,cell type:Whole embryo|developmental stage:18  24  36  48  72 hpf|strain:AB|genotype:ctrl cr  tbx16  tbx16 tbx16l  tbx16 msgn1  cdx4  cdx4 cdx1a  tbxta  tbx16  tbx16l  msgn1  wnt3a wnt8a  noto  smo  tbx1  hand2  egr2b  epha4a  hoxb1a  mafba  zc4h2  phox2a  foxi1  hgfa  met  foxd3  tfap2a  tfap2a foxd3,GSM6127483,GSM6127483: Zebrafish perturbation atlas plates4 6; Danio rerio; RNA Seq,GSM6127483,,1,Embryos were transferred  one by one  into separate wells of a 96 well V bottom plate containing 75μL of 1X TrypLE + 2 mg/mL Collagenase P. Embryos were then dissociated by manual trituration at 30℃ once every 5 minutes. Dissociation continued until no visible chunks were present under a dissecting scope  which took between 20 40 minutes depending on embryo stage e.g. 20 minutes for 18hpf and 40 minutes for 72hpf. Stop solution [1x dPBS  5% FBS ] was then added to each well to quench the proteases. three prime end capture of polyAdenylated transcripts. In situ reverse transcription with barcoded RT primers followed by second strand synthesis  tagmentation and index PCR gives each nucleus and all the RNA molecules contained within it  a unique combination of barcode sequences. These barcodes are then demultiplexed to create single cells in silico.,GEO Accession:GSM6127483,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina NovaSeq 6000,,SRP374541,,,GAP16_run2.1.fq.part1-L001.fastq.gz,fastq,9504654900.0,95046549.0,GSM6127483 r1,0:100,A:3269236251;C:1823488887;G:1923030699;T:2488567098;N:331965,100,,,,3269236251,1823488887,1923030699,2488567098,331965,SRX15222308,SRS12956194,SRA1418000,GEO,"Genome Sciences, University of Washington",1,0.77932,,0.456,,0.82077,,0.50409,,100,,B,,usable mapping rate,illumina,novaseq_era,3prime,random_priming,unknown,sc,single_cell_plate,scirnaseq,,United States,2022-05-10,Larval,Larval,Whole Organism,All anatomical structures
69694,SRR19155733,SRX15222308,SRS12956194,SRP374541,PRJNA836866,Embryo scale reverse genetics at single cell resolution,GSE202639,Transcriptome Analysis,The maturation of single cell transcriptomic technologies has facilitated the generation of comprehensive cellular atlases from whole embryos. A majority of this data  however  has been collected from wild type embryos without xxx appreciation for latent variation present in development. Here we present single cell transcriptomic data from 1812 individually resolved developing zebrafish embryos  encompassing 19 time points  23 genetic perturbations  and totaling 3.2M cells. The high degree of replication in our study 8 or more embryos per condition allows us to estimate the variance in cell type abundance organism wide and to detect perturbation dependent deviance in cell type composition relative to wild type embryos. Our approach is sensitive to rare cell types  resolving developmental trajectories and genetic dependencies in the cranial ganglia neurons  a cell population that comprises less than 1% of the embryo. Additionally  time series profiling of individual mutants identified a group of brachyury independent cells with strikingly similar transcriptomes to notochord sheath cells  leading to new hypotheses about the origins of the skull. We anticipate that standardized collection of high resolution  organism scale single cell data from large numbers of individual embryos will enable mapping the genetic dependencies of zebrafish cell types  while also addressing long standing challenges in developmental genetics  including the cellular and transcriptional plasticity underlying phenotypic diversity across individuals. Overall design: Whole zebrafish embryos were collected across multiple timepoints or in response to genetic perturbations with sci RNA seq3. Please note that the following processed data files have been updated on Mar 7  2023: zperturb full cell metadata.csv zperturb full gene metadata.csv zperturb full raw counts.RDS,,pubmed:37968389,,Zebrafish perturbation atlas plates4 6,GSM6127483,,tissue:Whole embryo|cell type:Whole embryo|developmental stage:18  24  36  48  72 hpf|strain:AB|genotype:ctrl cr  tbx16  tbx16 tbx16l  tbx16 msgn1  cdx4  cdx4 cdx1a  tbxta  tbx16  tbx16l  msgn1  wnt3a wnt8a  noto  smo  tbx1  hand2  egr2b  epha4a  hoxb1a  mafba  zc4h2  phox2a  foxi1  hgfa  met  foxd3  tfap2a  tfap2a foxd3,Zebrafish perturbation atlas plates4 6,The demultiplexing  barcoded processing  gene counting and aggregation were performed using the Brotman Baty sci RNA seq demultiplexing and processing pipelines https://github.com/bbi lab/bbi dmux; https://github.com/bbi lab/bbi sci. Count matrix was loaded into Monocle3 for further analysis Assembly: GRCz11 Supplementary files format and content: Count Matrix RDS file  Cell Metadata comma separated values  .csv  Gene Metadata comma separated values  .csv Supplementary files format and content: zperturb pilot cds.RDS monocle3 cell data set object Supplementary files format and content: zperturb pilot raw counts.RDS dgCMatrix with raw counts Supplementary files format and content: zperturb pilot cell metadata.csv cell metadata table comma separated Supplementary files format and content: zperturb pilot gene metadata.csv gene metadata table comma separated Supplementary files format and content: zperturb pilot hashTable.txt hash oligo count table Supplementary files format and content: reference cds.RDS monocle3 cell data set object Supplementary files format and content: reference raw counts.RDS dgCMatrix with raw counts Supplementary files format and content: reference cell metadata.csv cell metadata table comma separated Supplementary files format and content: reference gene metadata.csv gene metadata table comma separated Supplementary files format and content: reference expt1 hashTable.txt hash oligo count table Supplementary files format and content: reference expt2 hashTable.txt hash oligo count table Supplementary files format and content: reference expt3 hashTable.txt hash oligo count table Supplementary files format and content: zperturb full cds.RDS monocle3 cell data set object Supplementary files format and content: zperturb full raw counts.RDS dgCMatrix with raw counts Supplementary files format and content: zperturb full cell metadata.csv cell metadata table comma separated Supplementary files format and content: zperturb full gene metadata.csv gene metadata table comma separated Supplementary files format and content: zperturb full run1 hashTable.txt hash oligo count table Supplementary files format and content: zperturb full run2 hashTable.txt hash oligo count table,Whole embryo,,Embryos were transferred  one by one  into separate wells of a 96 well V bottom plate containing 75μL of 1X TrypLE + 2 mg/mL Collagenase P. Embryos were then dissociated by manual trituration at 30℃ once every 5 minutes. Dissociation continued until no visible chunks were present under a dissecting scope  which took between 20 40 minutes depending on embryo stage e.g. 20 minutes for 18hpf and 40 minutes for 72hpf. Stop solution [1x dPBS  5% FBS ] was then added to each well to quench the proteases. three prime end capture of polyAdenylated transcripts. In situ reverse transcription with barcoded RT primers followed by second strand synthesis  tagmentation and index PCR gives each nucleus and all the RNA molecules contained within it  a unique combination of barcode sequences. These barcodes are then demultiplexed to create single cells in silico.,Zebrafish embryos were raised in embryo media at 28.5C.,cell type:Whole embryo|developmental stage:18  24  36  48  72 hpf|strain:AB|genotype:ctrl cr  tbx16  tbx16 tbx16l  tbx16 msgn1  cdx4  cdx4 cdx1a  tbxta  tbx16  tbx16l  msgn1  wnt3a wnt8a  noto  smo  tbx1  hand2  egr2b  epha4a  hoxb1a  mafba  zc4h2  phox2a  foxi1  hgfa  met  foxd3  tfap2a  tfap2a foxd3,GSM6127483,GSM6127483: Zebrafish perturbation atlas plates4 6; Danio rerio; RNA Seq,GSM6127483,,1,Embryos were transferred  one by one  into separate wells of a 96 well V bottom plate containing 75μL of 1X TrypLE + 2 mg/mL Collagenase P. Embryos were then dissociated by manual trituration at 30℃ once every 5 minutes. Dissociation continued until no visible chunks were present under a dissecting scope  which took between 20 40 minutes depending on embryo stage e.g. 20 minutes for 18hpf and 40 minutes for 72hpf. Stop solution [1x dPBS  5% FBS ] was then added to each well to quench the proteases. three prime end capture of polyAdenylated transcripts. In situ reverse transcription with barcoded RT primers followed by second strand synthesis  tagmentation and index PCR gives each nucleus and all the RNA molecules contained within it  a unique combination of barcode sequences. These barcodes are then demultiplexed to create single cells in silico.,GEO Accession:GSM6127483,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina NovaSeq 6000,,SRP374541,,,GAP16_run2.1.fq.part3-L002.fastq.gz,fastq,8028928800.0,80289288.0,GSM6127483 r10,0:100,A:2759535999;C:1524680089;G:1624278378;T:2120102990;N:331344,100,,,,2759535999,1524680089,1624278378,2120102990,331344,SRX15222308,SRS12956194,SRA1418000,GEO,"Genome Sciences, University of Washington",1,0.77955,,0.45873,,0.81848,,0.50675,,100,,B,,usable mapping rate,illumina,novaseq_era,3prime,random_priming,unknown,sc,single_cell_plate,scirnaseq,,United States,2022-05-10,Larval,Larval,Whole Organism,All anatomical structures
69695,SRR19155734,SRX15222308,SRS12956194,SRP374541,PRJNA836866,Embryo scale reverse genetics at single cell resolution,GSE202639,Transcriptome Analysis,The maturation of single cell transcriptomic technologies has facilitated the generation of comprehensive cellular atlases from whole embryos. A majority of this data  however  has been collected from wild type embryos without xxx appreciation for latent variation present in development. Here we present single cell transcriptomic data from 1812 individually resolved developing zebrafish embryos  encompassing 19 time points  23 genetic perturbations  and totaling 3.2M cells. The high degree of replication in our study 8 or more embryos per condition allows us to estimate the variance in cell type abundance organism wide and to detect perturbation dependent deviance in cell type composition relative to wild type embryos. Our approach is sensitive to rare cell types  resolving developmental trajectories and genetic dependencies in the cranial ganglia neurons  a cell population that comprises less than 1% of the embryo. Additionally  time series profiling of individual mutants identified a group of brachyury independent cells with strikingly similar transcriptomes to notochord sheath cells  leading to new hypotheses about the origins of the skull. We anticipate that standardized collection of high resolution  organism scale single cell data from large numbers of individual embryos will enable mapping the genetic dependencies of zebrafish cell types  while also addressing long standing challenges in developmental genetics  including the cellular and transcriptional plasticity underlying phenotypic diversity across individuals. Overall design: Whole zebrafish embryos were collected across multiple timepoints or in response to genetic perturbations with sci RNA seq3. Please note that the following processed data files have been updated on Mar 7  2023: zperturb full cell metadata.csv zperturb full gene metadata.csv zperturb full raw counts.RDS,,pubmed:37968389,,Zebrafish perturbation atlas plates4 6,GSM6127483,,tissue:Whole embryo|cell type:Whole embryo|developmental stage:18  24  36  48  72 hpf|strain:AB|genotype:ctrl cr  tbx16  tbx16 tbx16l  tbx16 msgn1  cdx4  cdx4 cdx1a  tbxta  tbx16  tbx16l  msgn1  wnt3a wnt8a  noto  smo  tbx1  hand2  egr2b  epha4a  hoxb1a  mafba  zc4h2  phox2a  foxi1  hgfa  met  foxd3  tfap2a  tfap2a foxd3,Zebrafish perturbation atlas plates4 6,The demultiplexing  barcoded processing  gene counting and aggregation were performed using the Brotman Baty sci RNA seq demultiplexing and processing pipelines https://github.com/bbi lab/bbi dmux; https://github.com/bbi lab/bbi sci. Count matrix was loaded into Monocle3 for further analysis Assembly: GRCz11 Supplementary files format and content: Count Matrix RDS file  Cell Metadata comma separated values  .csv  Gene Metadata comma separated values  .csv Supplementary files format and content: zperturb pilot cds.RDS monocle3 cell data set object Supplementary files format and content: zperturb pilot raw counts.RDS dgCMatrix with raw counts Supplementary files format and content: zperturb pilot cell metadata.csv cell metadata table comma separated Supplementary files format and content: zperturb pilot gene metadata.csv gene metadata table comma separated Supplementary files format and content: zperturb pilot hashTable.txt hash oligo count table Supplementary files format and content: reference cds.RDS monocle3 cell data set object Supplementary files format and content: reference raw counts.RDS dgCMatrix with raw counts Supplementary files format and content: reference cell metadata.csv cell metadata table comma separated Supplementary files format and content: reference gene metadata.csv gene metadata table comma separated Supplementary files format and content: reference expt1 hashTable.txt hash oligo count table Supplementary files format and content: reference expt2 hashTable.txt hash oligo count table Supplementary files format and content: reference expt3 hashTable.txt hash oligo count table Supplementary files format and content: zperturb full cds.RDS monocle3 cell data set object Supplementary files format and content: zperturb full raw counts.RDS dgCMatrix with raw counts Supplementary files format and content: zperturb full cell metadata.csv cell metadata table comma separated Supplementary files format and content: zperturb full gene metadata.csv gene metadata table comma separated Supplementary files format and content: zperturb full run1 hashTable.txt hash oligo count table Supplementary files format and content: zperturb full run2 hashTable.txt hash oligo count table,Whole embryo,,Embryos were transferred  one by one  into separate wells of a 96 well V bottom plate containing 75μL of 1X TrypLE + 2 mg/mL Collagenase P. Embryos were then dissociated by manual trituration at 30℃ once every 5 minutes. Dissociation continued until no visible chunks were present under a dissecting scope  which took between 20 40 minutes depending on embryo stage e.g. 20 minutes for 18hpf and 40 minutes for 72hpf. Stop solution [1x dPBS  5% FBS ] was then added to each well to quench the proteases. three prime end capture of polyAdenylated transcripts. In situ reverse transcription with barcoded RT primers followed by second strand synthesis  tagmentation and index PCR gives each nucleus and all the RNA molecules contained within it  a unique combination of barcode sequences. These barcodes are then demultiplexed to create single cells in silico.,Zebrafish embryos were raised in embryo media at 28.5C.,cell type:Whole embryo|developmental stage:18  24  36  48  72 hpf|strain:AB|genotype:ctrl cr  tbx16  tbx16 tbx16l  tbx16 msgn1  cdx4  cdx4 cdx1a  tbxta  tbx16  tbx16l  msgn1  wnt3a wnt8a  noto  smo  tbx1  hand2  egr2b  epha4a  hoxb1a  mafba  zc4h2  phox2a  foxi1  hgfa  met  foxd3  tfap2a  tfap2a foxd3,GSM6127483,GSM6127483: Zebrafish perturbation atlas plates4 6; Danio rerio; RNA Seq,GSM6127483,,1,Embryos were transferred  one by one  into separate wells of a 96 well V bottom plate containing 75μL of 1X TrypLE + 2 mg/mL Collagenase P. Embryos were then dissociated by manual trituration at 30℃ once every 5 minutes. Dissociation continued until no visible chunks were present under a dissecting scope  which took between 20 40 minutes depending on embryo stage e.g. 20 minutes for 18hpf and 40 minutes for 72hpf. Stop solution [1x dPBS  5% FBS ] was then added to each well to quench the proteases. three prime end capture of polyAdenylated transcripts. In situ reverse transcription with barcoded RT primers followed by second strand synthesis  tagmentation and index PCR gives each nucleus and all the RNA molecules contained within it  a unique combination of barcode sequences. These barcodes are then demultiplexed to create single cells in silico.,GEO Accession:GSM6127483,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina NovaSeq 6000,,SRP374541,,,GAP16_run2.1.fq.part3-L003.fastq.gz,fastq,7761212200.0,77612122.0,GSM6127483 r11,0:100,A:2688948978;C:1462620928;G:1562357644;T:2046943755;N:340895,100,,,,2688948978,1462620928,1562357644,2046943755,340895,SRX15222308,SRS12956194,SRA1418000,GEO,"Genome Sciences, University of Washington",1,0.77683,,0.45932,,0.82122,,0.50551,,100,,B,,usable mapping rate,illumina,novaseq_era,3prime,random_priming,unknown,sc,single_cell_plate,scirnaseq,,United States,2022-05-10,Larval,Larval,Whole Organism,All anatomical structures
69696,SRR19155735,SRX15222308,SRS12956194,SRP374541,PRJNA836866,Embryo scale reverse genetics at single cell resolution,GSE202639,Transcriptome Analysis,The maturation of single cell transcriptomic technologies has facilitated the generation of comprehensive cellular atlases from whole embryos. A majority of this data  however  has been collected from wild type embryos without xxx appreciation for latent variation present in development. Here we present single cell transcriptomic data from 1812 individually resolved developing zebrafish embryos  encompassing 19 time points  23 genetic perturbations  and totaling 3.2M cells. The high degree of replication in our study 8 or more embryos per condition allows us to estimate the variance in cell type abundance organism wide and to detect perturbation dependent deviance in cell type composition relative to wild type embryos. Our approach is sensitive to rare cell types  resolving developmental trajectories and genetic dependencies in the cranial ganglia neurons  a cell population that comprises less than 1% of the embryo. Additionally  time series profiling of individual mutants identified a group of brachyury independent cells with strikingly similar transcriptomes to notochord sheath cells  leading to new hypotheses about the origins of the skull. We anticipate that standardized collection of high resolution  organism scale single cell data from large numbers of individual embryos will enable mapping the genetic dependencies of zebrafish cell types  while also addressing long standing challenges in developmental genetics  including the cellular and transcriptional plasticity underlying phenotypic diversity across individuals. Overall design: Whole zebrafish embryos were collected across multiple timepoints or in response to genetic perturbations with sci RNA seq3. Please note that the following processed data files have been updated on Mar 7  2023: zperturb full cell metadata.csv zperturb full gene metadata.csv zperturb full raw counts.RDS,,pubmed:37968389,,Zebrafish perturbation atlas plates4 6,GSM6127483,,tissue:Whole embryo|cell type:Whole embryo|developmental stage:18  24  36  48  72 hpf|strain:AB|genotype:ctrl cr  tbx16  tbx16 tbx16l  tbx16 msgn1  cdx4  cdx4 cdx1a  tbxta  tbx16  tbx16l  msgn1  wnt3a wnt8a  noto  smo  tbx1  hand2  egr2b  epha4a  hoxb1a  mafba  zc4h2  phox2a  foxi1  hgfa  met  foxd3  tfap2a  tfap2a foxd3,Zebrafish perturbation atlas plates4 6,The demultiplexing  barcoded processing  gene counting and aggregation were performed using the Brotman Baty sci RNA seq demultiplexing and processing pipelines https://github.com/bbi lab/bbi dmux; https://github.com/bbi lab/bbi sci. Count matrix was loaded into Monocle3 for further analysis Assembly: GRCz11 Supplementary files format and content: Count Matrix RDS file  Cell Metadata comma separated values  .csv  Gene Metadata comma separated values  .csv Supplementary files format and content: zperturb pilot cds.RDS monocle3 cell data set object Supplementary files format and content: zperturb pilot raw counts.RDS dgCMatrix with raw counts Supplementary files format and content: zperturb pilot cell metadata.csv cell metadata table comma separated Supplementary files format and content: zperturb pilot gene metadata.csv gene metadata table comma separated Supplementary files format and content: zperturb pilot hashTable.txt hash oligo count table Supplementary files format and content: reference cds.RDS monocle3 cell data set object Supplementary files format and content: reference raw counts.RDS dgCMatrix with raw counts Supplementary files format and content: reference cell metadata.csv cell metadata table comma separated Supplementary files format and content: reference gene metadata.csv gene metadata table comma separated Supplementary files format and content: reference expt1 hashTable.txt hash oligo count table Supplementary files format and content: reference expt2 hashTable.txt hash oligo count table Supplementary files format and content: reference expt3 hashTable.txt hash oligo count table Supplementary files format and content: zperturb full cds.RDS monocle3 cell data set object Supplementary files format and content: zperturb full raw counts.RDS dgCMatrix with raw counts Supplementary files format and content: zperturb full cell metadata.csv cell metadata table comma separated Supplementary files format and content: zperturb full gene metadata.csv gene metadata table comma separated Supplementary files format and content: zperturb full run1 hashTable.txt hash oligo count table Supplementary files format and content: zperturb full run2 hashTable.txt hash oligo count table,Whole embryo,,Embryos were transferred  one by one  into separate wells of a 96 well V bottom plate containing 75μL of 1X TrypLE + 2 mg/mL Collagenase P. Embryos were then dissociated by manual trituration at 30℃ once every 5 minutes. Dissociation continued until no visible chunks were present under a dissecting scope  which took between 20 40 minutes depending on embryo stage e.g. 20 minutes for 18hpf and 40 minutes for 72hpf. Stop solution [1x dPBS  5% FBS ] was then added to each well to quench the proteases. three prime end capture of polyAdenylated transcripts. In situ reverse transcription with barcoded RT primers followed by second strand synthesis  tagmentation and index PCR gives each nucleus and all the RNA molecules contained within it  a unique combination of barcode sequences. These barcodes are then demultiplexed to create single cells in silico.,Zebrafish embryos were raised in embryo media at 28.5C.,cell type:Whole embryo|developmental stage:18  24  36  48  72 hpf|strain:AB|genotype:ctrl cr  tbx16  tbx16 tbx16l  tbx16 msgn1  cdx4  cdx4 cdx1a  tbxta  tbx16  tbx16l  msgn1  wnt3a wnt8a  noto  smo  tbx1  hand2  egr2b  epha4a  hoxb1a  mafba  zc4h2  phox2a  foxi1  hgfa  met  foxd3  tfap2a  tfap2a foxd3,GSM6127483,GSM6127483: Zebrafish perturbation atlas plates4 6; Danio rerio; RNA Seq,GSM6127483,,1,Embryos were transferred  one by one  into separate wells of a 96 well V bottom plate containing 75μL of 1X TrypLE + 2 mg/mL Collagenase P. Embryos were then dissociated by manual trituration at 30℃ once every 5 minutes. Dissociation continued until no visible chunks were present under a dissecting scope  which took between 20 40 minutes depending on embryo stage e.g. 20 minutes for 18hpf and 40 minutes for 72hpf. Stop solution [1x dPBS  5% FBS ] was then added to each well to quench the proteases. three prime end capture of polyAdenylated transcripts. In situ reverse transcription with barcoded RT primers followed by second strand synthesis  tagmentation and index PCR gives each nucleus and all the RNA molecules contained within it  a unique combination of barcode sequences. These barcodes are then demultiplexed to create single cells in silico.,GEO Accession:GSM6127483,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina NovaSeq 6000,,SRP374541,,,GAP16_run2.1.fq.part3-L004.fastq.gz,fastq,7818788300.0,78187883.0,GSM6127483 r12,0:100,A:2710591219;C:1472629959;G:1572332337;T:2062881445;N:353340,100,,,,2710591219,1472629959,1572332337,2062881445,353340,SRX15222308,SRS12956194,SRA1418000,GEO,"Genome Sciences, University of Washington",1,0.77581,,0.45743,,0.81858,,0.50651,,100,,B,,usable mapping rate,illumina,novaseq_era,3prime,random_priming,unknown,sc,single_cell_plate,scirnaseq,,United States,2022-05-10,Larval,Larval,Whole Organism,All anatomical structures
69697,SRR19155736,SRX15222308,SRS12956194,SRP374541,PRJNA836866,Embryo scale reverse genetics at single cell resolution,GSE202639,Transcriptome Analysis,The maturation of single cell transcriptomic technologies has facilitated the generation of comprehensive cellular atlases from whole embryos. A majority of this data  however  has been collected from wild type embryos without xxx appreciation for latent variation present in development. Here we present single cell transcriptomic data from 1812 individually resolved developing zebrafish embryos  encompassing 19 time points  23 genetic perturbations  and totaling 3.2M cells. The high degree of replication in our study 8 or more embryos per condition allows us to estimate the variance in cell type abundance organism wide and to detect perturbation dependent deviance in cell type composition relative to wild type embryos. Our approach is sensitive to rare cell types  resolving developmental trajectories and genetic dependencies in the cranial ganglia neurons  a cell population that comprises less than 1% of the embryo. Additionally  time series profiling of individual mutants identified a group of brachyury independent cells with strikingly similar transcriptomes to notochord sheath cells  leading to new hypotheses about the origins of the skull. We anticipate that standardized collection of high resolution  organism scale single cell data from large numbers of individual embryos will enable mapping the genetic dependencies of zebrafish cell types  while also addressing long standing challenges in developmental genetics  including the cellular and transcriptional plasticity underlying phenotypic diversity across individuals. Overall design: Whole zebrafish embryos were collected across multiple timepoints or in response to genetic perturbations with sci RNA seq3. Please note that the following processed data files have been updated on Mar 7  2023: zperturb full cell metadata.csv zperturb full gene metadata.csv zperturb full raw counts.RDS,,pubmed:37968389,,Zebrafish perturbation atlas plates4 6,GSM6127483,,tissue:Whole embryo|cell type:Whole embryo|developmental stage:18  24  36  48  72 hpf|strain:AB|genotype:ctrl cr  tbx16  tbx16 tbx16l  tbx16 msgn1  cdx4  cdx4 cdx1a  tbxta  tbx16  tbx16l  msgn1  wnt3a wnt8a  noto  smo  tbx1  hand2  egr2b  epha4a  hoxb1a  mafba  zc4h2  phox2a  foxi1  hgfa  met  foxd3  tfap2a  tfap2a foxd3,Zebrafish perturbation atlas plates4 6,The demultiplexing  barcoded processing  gene counting and aggregation were performed using the Brotman Baty sci RNA seq demultiplexing and processing pipelines https://github.com/bbi lab/bbi dmux; https://github.com/bbi lab/bbi sci. Count matrix was loaded into Monocle3 for further analysis Assembly: GRCz11 Supplementary files format and content: Count Matrix RDS file  Cell Metadata comma separated values  .csv  Gene Metadata comma separated values  .csv Supplementary files format and content: zperturb pilot cds.RDS monocle3 cell data set object Supplementary files format and content: zperturb pilot raw counts.RDS dgCMatrix with raw counts Supplementary files format and content: zperturb pilot cell metadata.csv cell metadata table comma separated Supplementary files format and content: zperturb pilot gene metadata.csv gene metadata table comma separated Supplementary files format and content: zperturb pilot hashTable.txt hash oligo count table Supplementary files format and content: reference cds.RDS monocle3 cell data set object Supplementary files format and content: reference raw counts.RDS dgCMatrix with raw counts Supplementary files format and content: reference cell metadata.csv cell metadata table comma separated Supplementary files format and content: reference gene metadata.csv gene metadata table comma separated Supplementary files format and content: reference expt1 hashTable.txt hash oligo count table Supplementary files format and content: reference expt2 hashTable.txt hash oligo count table Supplementary files format and content: reference expt3 hashTable.txt hash oligo count table Supplementary files format and content: zperturb full cds.RDS monocle3 cell data set object Supplementary files format and content: zperturb full raw counts.RDS dgCMatrix with raw counts Supplementary files format and content: zperturb full cell metadata.csv cell metadata table comma separated Supplementary files format and content: zperturb full gene metadata.csv gene metadata table comma separated Supplementary files format and content: zperturb full run1 hashTable.txt hash oligo count table Supplementary files format and content: zperturb full run2 hashTable.txt hash oligo count table,Whole embryo,,Embryos were transferred  one by one  into separate wells of a 96 well V bottom plate containing 75μL of 1X TrypLE + 2 mg/mL Collagenase P. Embryos were then dissociated by manual trituration at 30℃ once every 5 minutes. Dissociation continued until no visible chunks were present under a dissecting scope  which took between 20 40 minutes depending on embryo stage e.g. 20 minutes for 18hpf and 40 minutes for 72hpf. Stop solution [1x dPBS  5% FBS ] was then added to each well to quench the proteases. three prime end capture of polyAdenylated transcripts. In situ reverse transcription with barcoded RT primers followed by second strand synthesis  tagmentation and index PCR gives each nucleus and all the RNA molecules contained within it  a unique combination of barcode sequences. These barcodes are then demultiplexed to create single cells in silico.,Zebrafish embryos were raised in embryo media at 28.5C.,cell type:Whole embryo|developmental stage:18  24  36  48  72 hpf|strain:AB|genotype:ctrl cr  tbx16  tbx16 tbx16l  tbx16 msgn1  cdx4  cdx4 cdx1a  tbxta  tbx16  tbx16l  msgn1  wnt3a wnt8a  noto  smo  tbx1  hand2  egr2b  epha4a  hoxb1a  mafba  zc4h2  phox2a  foxi1  hgfa  met  foxd3  tfap2a  tfap2a foxd3,GSM6127483,GSM6127483: Zebrafish perturbation atlas plates4 6; Danio rerio; RNA Seq,GSM6127483,,1,Embryos were transferred  one by one  into separate wells of a 96 well V bottom plate containing 75μL of 1X TrypLE + 2 mg/mL Collagenase P. Embryos were then dissociated by manual trituration at 30℃ once every 5 minutes. Dissociation continued until no visible chunks were present under a dissecting scope  which took between 20 40 minutes depending on embryo stage e.g. 20 minutes for 18hpf and 40 minutes for 72hpf. Stop solution [1x dPBS  5% FBS ] was then added to each well to quench the proteases. three prime end capture of polyAdenylated transcripts. In situ reverse transcription with barcoded RT primers followed by second strand synthesis  tagmentation and index PCR gives each nucleus and all the RNA molecules contained within it  a unique combination of barcode sequences. These barcodes are then demultiplexed to create single cells in silico.,GEO Accession:GSM6127483,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina NovaSeq 6000,,SRP374541,,,GAP16_run2.2.fq.part1-L001.fastq.gz,fastq,8677521200.0,86775212.0,GSM6127483 r13,0:100,A:3001182398;C:1650845100;G:1747702021;T:2277489351;N:302330,100,,,,3001182398,1650845100,1747702021,2277489351,302330,SRX15222308,SRS12956194,SRA1418000,GEO,"Genome Sciences, University of Washington",1,0.77731,,0.45744,,0.82205,,0.50398,,100,,B,,usable mapping rate,illumina,novaseq_era,3prime,random_priming,unknown,sc,single_cell_plate,scirnaseq,,United States,2022-05-10,Larval,Larval,Whole Organism,All anatomical structures