metadata: run_metadata: 95 rows where experiment.library_source = "TRANSCRIPTOMIC", experiment.library_strategy = "OTHER" and technology = "scirnaseq"

95 rows where experiment.library_source = "TRANSCRIPTOMIC", experiment.library_strategy = "OTHER" and technology = "scirnaseq"

descending

Link	rowid ▼	run.accession	experiment.accession	sample.accession	study.accession	bioproject	study.title	study.alias	study.type	study.abstract	sample.title	sample.alias	sample.attributes	GEOsample.title	GEOsample.dataprocessing	GEOsample.source	GEOsample.extractprotocol	GEOsample.growthprotocol	GEOsample.characteristics	GEOsample.accession	experiment.title	experiment.alias	experiment.library_name	experiment.design_description	experiment.library_construction_protocol	experiment.library_strategy	experiment.library_source	experiment.library_selection	experiment.library_layout	experiment.platform	experiment.instrument_model	experiment.study_ref	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.Acount	run.Ccount	run.Gcount	run.Tcount	run.Ncount	run.experiment	run.pool_member	submission.accession	submission.srasource	submission.bioprojectsource	seqdetective.judgement.mate1	seqdetective.judgement.reason	platform_family	instrument_generation	read_bias	selection_class	prep_kit	sc_or_bulk	tech_class	technology	submission.bioprojectsource.country	earliest_date	devstage_curation	devstage_curation_coarse	tissue_curation	tissue_curation_coarse
34712	34712	SRR32232924	SRX27573024	SRS23984677	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 05H	GSM8775334	source name:6 dpf head\|tissue:6 dpf head\|well:P1 H05\|rtindex:TTCGCCTCCA\|genotype:GAL4 replicate2\|geo loc name:missing\|collection date:missing	Well P1 05H	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 H05\|rtindex:TTCGCCTCCA\|genotype:GAL4 replicate2	GSM8775334	GSM8775334: Well P1 05H; Danio rerio; OTHER	GSM8775334 r1	GSM8775334	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-05H.R2.fastq.gz P1-05H_S40_I1_001.fastq.gz P1-05H.R1.fastq.gz	fastq fastq fastq	828.0	9.0	GSM8775334 r1	0:34 1:48 2:10	A:210;C:74;G:47;T:101;N:0	34	48	10	210	74	47	101	0	SRX27573024	SRS23984677	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34713	34713	SRR32232925	SRX27573023	SRS23984676	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 05G	GSM8775333	source name:6 dpf head\|tissue:6 dpf head\|well:P1 G05\|rtindex:TTACCTCGAC\|genotype:GAL4 replicate2\|geo loc name:missing\|collection date:missing	Well P1 05G	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 G05\|rtindex:TTACCTCGAC\|genotype:GAL4 replicate2	GSM8775333	GSM8775333: Well P1 05G; Danio rerio; OTHER	GSM8775333 r1	GSM8775333	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-05G.R1.fastq.gz P1-05G.R2.fastq.gz P1-05G_S39_I1_001.fastq.gz	fastq fastq fastq	3345856.0	36368.0	GSM8775333 r1	0:34 1:48 2:10	A:602824;C:246794;G:422064;T:471661;N:2321	34	48	10	602824	246794	422064	471661	2321	SRX27573023	SRS23984676	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34714	34714	SRR32232926	SRX27573022	SRS23984675	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 05F	GSM8775332	source name:6 dpf head\|tissue:6 dpf head\|well:P1 F05\|rtindex:CCATTATCTA\|genotype:GAL4 replicate2\|geo loc name:missing\|collection date:missing	Well P1 05F	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 F05\|rtindex:CCATTATCTA\|genotype:GAL4 replicate2	GSM8775332	GSM8775332: Well P1 05F; Danio rerio; OTHER	GSM8775332 r1	GSM8775332	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-05F.R1.fastq.gz P1-05F.R2.fastq.gz P1-05F_S38_I1_001.fastq.gz	fastq fastq fastq	143353940.0	1558195.0	GSM8775332 r1	0:34 1:48 2:10	A:32466011;C:9391164;G:14603239;T:18226855;N:106091	34	48	10	32466011	9391164	14603239	18226855	106091	SRX27573022	SRS23984675	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34715	34715	SRR32232927	SRX27573021	SRS23984674	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 05E	GSM8775331	source name:6 dpf head\|tissue:6 dpf head\|well:P1 E05\|rtindex:CTAGTAGTCT\|genotype:GAL4 replicate2\|geo loc name:missing\|collection date:missing	Well P1 05E	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 E05\|rtindex:CTAGTAGTCT\|genotype:GAL4 replicate2	GSM8775331	GSM8775331: Well P1 05E; Danio rerio; OTHER	GSM8775331 r1	GSM8775331	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-05E.R1.fastq.gz P1-05E.R2.fastq.gz P1-05E_S37_I1_001.fastq.gz	fastq fastq fastq	45118824.0	490422.0	GSM8775331 r1	0:34 1:48 2:10	A:7745921;C:3203071;G:7199342;T:5360465;N:31457	34	48	10	7745921	3203071	7199342	5360465	31457	SRX27573021	SRS23984674	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34716	34716	SRR32232928	SRX27573020	SRS23984673	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 05D	GSM8775330	source name:6 dpf head\|tissue:6 dpf head\|well:P1 D05\|rtindex:AGAGAAGGTT\|genotype:GAL4 replicate2\|geo loc name:missing\|collection date:missing	Well P1 05D	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 D05\|rtindex:AGAGAAGGTT\|genotype:GAL4 replicate2	GSM8775330	GSM8775330: Well P1 05D; Danio rerio; OTHER	GSM8775330 r1	GSM8775330	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-05D.R1.fastq.gz P1-05D.R2.fastq.gz P1-05D_S36_I1_001.fastq.gz	fastq fastq fastq	194869248.0	2118144.0	GSM8775330 r1	0:34 1:48 2:10	A:44069791;C:12867012;G:18935172;T:25657047;N:141890	34	48	10	44069791	12867012	18935172	25657047	141890	SRX27573020	SRS23984673	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34717	34717	SRR32232929	SRX27573019	SRS23984672	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 05C	GSM8775329	source name:6 dpf head\|tissue:6 dpf head\|well:P1 C05\|rtindex:TAGAGTAATA\|genotype:GAL4 replicate2\|geo loc name:missing\|collection date:missing	Well P1 05C	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 C05\|rtindex:TAGAGTAATA\|genotype:GAL4 replicate2	GSM8775329	GSM8775329: Well P1 05C; Danio rerio; OTHER	GSM8775329 r1	GSM8775329	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-05C.R1.fastq.gz P1-05C.R2.fastq.gz P1-05C_S35_I1_001.fastq.gz	fastq fastq fastq	47118352.0	512156.0	GSM8775329 r1	0:34 1:48 2:10	A:9098221;C:3194198;G:6516993;T:5738665;N:35411	34	48	10	9098221	3194198	6516993	5738665	35411	SRX27573019	SRS23984672	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34718	34718	SRR32232930	SRX27573018	SRS23984671	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 05B	GSM8775328	source name:6 dpf head\|tissue:6 dpf head\|well:P1 B05\|rtindex:CTTGCGCCGC\|genotype:GAL4 replicate2\|geo loc name:missing\|collection date:missing	Well P1 05B	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 B05\|rtindex:CTTGCGCCGC\|genotype:GAL4 replicate2	GSM8775328	GSM8775328: Well P1 05B; Danio rerio; OTHER	GSM8775328 r1	GSM8775328	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-05B.R1.fastq.gz P1-05B.R2.fastq.gz P1-05B_S34_I1_001.fastq.gz	fastq fastq fastq	664471748.0	7222519.0	GSM8775328 r1	0:34 1:48 2:10	A:146424611;C:46287832;G:64801496;T:88677718;N:489255	34	48	10	146424611	46287832	64801496	88677718	489255	SRX27573018	SRS23984671	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34719	34719	SRR32232931	SRX27573017	SRS23984670	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 05A	GSM8775327	source name:6 dpf head\|tissue:6 dpf head\|well:P1 A05\|rtindex:CCGAGAATCC\|genotype:GAL4 replicate2\|geo loc name:missing\|collection date:missing	Well P1 05A	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 A05\|rtindex:CCGAGAATCC\|genotype:GAL4 replicate2	GSM8775327	GSM8775327: Well P1 05A; Danio rerio; OTHER	GSM8775327 r1	GSM8775327	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-05A.R1.fastq.gz P1-05A.R2.fastq.gz P1-05A_S33_I1_001.fastq.gz	fastq fastq fastq	33378152.0	362806.0	GSM8775327 r1	0:34 1:48 2:10	A:5712677;C:2316258;G:5388122;T:3975276;N:22355	34	48	10	5712677	2316258	5388122	3975276	22355	SRX27573017	SRS23984670	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34720	34720	SRR32232932	SRX27573016	SRS23984669	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 12H	GSM8775389	source name:6 dpf head\|tissue:6 dpf head\|well:P1 H12\|rtindex:TAACTGGTTA\|genotype:UAS replicate3\|geo loc name:missing\|collection date:missing	Well P1 12H	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 H12\|rtindex:TAACTGGTTA\|genotype:UAS replicate3	GSM8775389	GSM8775389: Well P1 12H; Danio rerio; OTHER	GSM8775389 r1	GSM8775389	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-12H.R1.fastq.gz P1-12H.R2.fastq.gz P1-12H_S96_I1_001.fastq.gz	fastq fastq fastq	368.0	4.0	GSM8775389 r1	0:34 1:48 2:10	A:70;C:25;G:61;T:36;N:0	34	48	10	70	25	61	36	0	SRX27573016	SRS23984669	SRA2070568	UMass Chan	UMass Chan	T	under 1.2% mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34721	34721	SRR32232933	SRX27573015	SRS23984668	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 12G	GSM8775388	source name:6 dpf head\|tissue:6 dpf head\|well:P1 G12\|rtindex:GGTACCGGCA\|genotype:UAS replicate3\|geo loc name:missing\|collection date:missing	Well P1 12G	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 G12\|rtindex:GGTACCGGCA\|genotype:UAS replicate3	GSM8775388	GSM8775388: Well P1 12G; Danio rerio; OTHER	GSM8775388 r1	GSM8775388	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-12G.R1.fastq.gz P1-12G.R2.fastq.gz P1-12G_S95_I1_001.fastq.gz	fastq fastq fastq	11776.0	128.0	GSM8775388 r1	0:34 1:48 2:10	A:2531;C:932;G:1152;T:1529;N:0	34	48	10	2531	932	1152	1529	0	SRX27573015	SRS23984668	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34722	34722	SRR32232934	SRX27573014	SRS23984666	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 12F	GSM8775387	source name:6 dpf head\|tissue:6 dpf head\|well:P1 F12\|rtindex:CATTCGACGG\|genotype:UAS replicate3\|geo loc name:missing\|collection date:missing	Well P1 12F	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 F12\|rtindex:CATTCGACGG\|genotype:UAS replicate3	GSM8775387	GSM8775387: Well P1 12F; Danio rerio; OTHER	GSM8775387 r1	GSM8775387	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-12F.R1.fastq.gz P1-12F.R2.fastq.gz P1-12F_S94_I1_001.fastq.gz	fastq fastq fastq	1012.0	11.0	GSM8775387 r1	0:34 1:48 2:10	A:192;C:67;G:130;T:139;N:0	34	48	10	192	67	130	139	0	SRX27573014	SRS23984666	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34723	34723	SRR32232935	SRX27573013	SRS23984667	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 12E	GSM8775386	source name:6 dpf head\|tissue:6 dpf head\|well:P1 E12\|rtindex:AACCATTGGA\|genotype:UAS replicate3\|geo loc name:missing\|collection date:missing	Well P1 12E	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 E12\|rtindex:AACCATTGGA\|genotype:UAS replicate3	GSM8775386	GSM8775386: Well P1 12E; Danio rerio; OTHER	GSM8775386 r1	GSM8775386	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-12E.R1.fastq.gz P1-12E.R2.fastq.gz P1-12E_S93_I1_001.fastq.gz	fastq fastq fastq	512348.0	5569.0	GSM8775386 r1	0:34 1:48 2:10	A:84224;C:34420;G:85992;T:62126;N:550	34	48	10	84224	34420	85992	62126	550	SRX27573013	SRS23984667	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34724	34724	SRR32232936	SRX27573012	SRS23984665	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 12D	GSM8775385	source name:6 dpf head\|tissue:6 dpf head\|well:P1 D12\|rtindex:CGAACGCCGG\|genotype:UAS replicate3\|geo loc name:missing\|collection date:missing	Well P1 12D	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 D12\|rtindex:CGAACGCCGG\|genotype:UAS replicate3	GSM8775385	GSM8775385: Well P1 12D; Danio rerio; OTHER	GSM8775385 r1	GSM8775385	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-12D.R1.fastq.gz P1-12D.R2.fastq.gz P1-12D_S92_I1_001.fastq.gz	fastq fastq fastq	14633428.0	159059.0	GSM8775385 r1	0:34 1:48 2:10	A:2133794;C:938571;G:2850027;T:1703116;N:9324	34	48	10	2133794	938571	2850027	1703116	9324	SRX27573012	SRS23984665	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34725	34725	SRR32232937	SRX27573011	SRS23984664	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 12C	GSM8775384	source name:6 dpf head\|tissue:6 dpf head\|well:P1 C12\|rtindex:GACCTTGATA\|genotype:UAS replicate3\|geo loc name:missing\|collection date:missing	Well P1 12C	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 C12\|rtindex:GACCTTGATA\|genotype:UAS replicate3	GSM8775384	GSM8775384: Well P1 12C; Danio rerio; OTHER	GSM8775384 r1	GSM8775384	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-12C.R1.fastq.gz P1-12C.R2.fastq.gz P1-12C_S91_I1_001.fastq.gz	fastq fastq fastq	24830524.0	269897.0	GSM8775384 r1	0:34 1:48 2:10	A:3753240;C:1715145;G:4579242;T:2889684;N:17745	34	48	10	3753240	1715145	4579242	2889684	17745	SRX27573011	SRS23984664	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34726	34726	SRR32232938	SRX27573010	SRS23984663	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 12B	GSM8775383	source name:6 dpf head\|tissue:6 dpf head\|well:P1 B12\|rtindex:TGGTCAGCCA\|genotype:UAS replicate3\|geo loc name:missing\|collection date:missing	Well P1 12B	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 B12\|rtindex:TGGTCAGCCA\|genotype:UAS replicate3	GSM8775383	GSM8775383: Well P1 12B; Danio rerio; OTHER	GSM8775383 r1	GSM8775383	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-12B.R1.fastq.gz P1-12B.R2.fastq.gz P1-12B_S90_I1_001.fastq.gz	fastq fastq fastq	22899628.0	248909.0	GSM8775383 r1	0:34 1:48 2:10	A:3385725;C:1544608;G:4301274;T:2699465;N:16560	34	48	10	3385725	1544608	4301274	2699465	16560	SRX27573010	SRS23984663	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34727	34727	SRR32232939	SRX27573009	SRS23984662	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 08H	GSM8775358	source name:6 dpf head\|tissue:6 dpf head\|well:P1 H08\|rtindex:AGCTCTCCTC\|genotype:UAS replicate2\|geo loc name:missing\|collection date:missing	Well P1 08H	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 H08\|rtindex:AGCTCTCCTC\|genotype:UAS replicate2	GSM8775358	GSM8775358: Well P1 08H; Danio rerio; OTHER	GSM8775358 r1	GSM8775358	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-08H.R1.fastq.gz P1-08H.R2.fastq.gz P1-08H_S64_I1_001.fastq.gz	fastq fastq fastq	552.0	6.0	GSM8775358 r1	0:34 1:48 2:10	A:114;C:44;G:65;T:65;N:0	34	48	10	114	44	65	65	0	SRX27573009	SRS23984662	SRA2070568	UMass Chan	UMass Chan	T	under 1.2% mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34728	34728	SRR32232940	SRX27573008	SRS23984661	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 08G	GSM8775357	source name:6 dpf head\|tissue:6 dpf head\|well:P1 G08\|rtindex:CGGTCAAGAA\|genotype:UAS replicate2\|geo loc name:missing\|collection date:missing	Well P1 08G	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 G08\|rtindex:CGGTCAAGAA\|genotype:UAS replicate2	GSM8775357	GSM8775357: Well P1 08G; Danio rerio; OTHER	GSM8775357 r1	GSM8775357	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-08G.R1.fastq.gz P1-08G.R2.fastq.gz P1-08G_S63_I1_001.fastq.gz	fastq fastq fastq	48348484.0	525527.0	GSM8775357 r1	0:34 1:48 2:10	A:8706380;C:3262708;G:7330788;T:5889373;N:36047	34	48	10	8706380	3262708	7330788	5889373	36047	SRX27573008	SRS23984661	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34729	34729	SRR32232941	SRX27573007	SRS23984660	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 08F	GSM8775356	source name:6 dpf head\|tissue:6 dpf head\|well:P1 F08\|rtindex:GATCGCTTCT\|genotype:UAS replicate2\|geo loc name:missing\|collection date:missing	Well P1 08F	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 F08\|rtindex:GATCGCTTCT\|genotype:UAS replicate2	GSM8775356	GSM8775356: Well P1 08F; Danio rerio; OTHER	GSM8775356 r1	GSM8775356	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-08F.R1.fastq.gz P1-08F.R2.fastq.gz P1-08F_S62_I1_001.fastq.gz	fastq fastq fastq	55386116.0	602023.0	GSM8775356 r1	0:34 1:48 2:10	A:10289406;C:3851867;G:8139616;T:6575750;N:40465	34	48	10	10289406	3851867	8139616	6575750	40465	SRX27573007	SRS23984660	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34730	34730	SRR32232942	SRX27573006	SRS23984659	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 08E	GSM8775355	source name:6 dpf head\|tissue:6 dpf head\|well:P1 E08\|rtindex:AACTAGGCGC\|genotype:UAS replicate2\|geo loc name:missing\|collection date:missing	Well P1 08E	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 E08\|rtindex:AACTAGGCGC\|genotype:UAS replicate2	GSM8775355	GSM8775355: Well P1 08E; Danio rerio; OTHER	GSM8775355 r1	GSM8775355	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-08E.R1.fastq.gz P1-08E.R2.fastq.gz P1-08E_S61_I1_001.fastq.gz	fastq fastq fastq	40465832.0	439846.0	GSM8775355 r1	0:34 1:48 2:10	A:6595819;C:2847582;G:6802285;T:4838399;N:28523	34	48	10	6595819	2847582	6802285	4838399	28523	SRX27573006	SRS23984659	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34731	34731	SRR32232943	SRX27573005	SRS23984658	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 08D	GSM8775354	source name:6 dpf head\|tissue:6 dpf head\|well:P1 D08\|rtindex:GGCTGAGCTC\|genotype:UAS replicate2\|geo loc name:missing\|collection date:missing	Well P1 08D	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 D08\|rtindex:GGCTGAGCTC\|genotype:UAS replicate2	GSM8775354	GSM8775354: Well P1 08D; Danio rerio; OTHER	GSM8775354 r1	GSM8775354	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-08D.R1.fastq.gz P1-08D.R2.fastq.gz P1-08D_S60_I1_001.fastq.gz	fastq fastq fastq	33437492.0	363451.0	GSM8775354 r1	0:34 1:48 2:10	A:4392257;C:1994475;G:7440459;T:3596074;N:22383	34	48	10	4392257	1994475	7440459	3596074	22383	SRX27573005	SRS23984658	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34732	34732	SRR32232944	SRX27573004	SRS23984656	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 08C	GSM8775353	source name:6 dpf head\|tissue:6 dpf head\|well:P1 C08\|rtindex:AGAACGTCTC\|genotype:UAS replicate2\|geo loc name:missing\|collection date:missing	Well P1 08C	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 C08\|rtindex:AGAACGTCTC\|genotype:UAS replicate2	GSM8775353	GSM8775353: Well P1 08C; Danio rerio; OTHER	GSM8775353 r1	GSM8775353	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-08C.R1.fastq.gz P1-08C.R2.fastq.gz P1-08C_S59_I1_001.fastq.gz	fastq fastq fastq	29029864.0	315542.0	GSM8775353 r1	0:34 1:48 2:10	A:4629476;C:2009722;G:5052427;T:3434235;N:20156	34	48	10	4629476	2009722	5052427	3434235	20156	SRX27573004	SRS23984656	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34733	34733	SRR32232945	SRX27573003	SRS23984657	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 08B	GSM8775352	source name:6 dpf head\|tissue:6 dpf head\|well:P1 B08\|rtindex:GCCATTCTCC\|genotype:UAS replicate2\|geo loc name:missing\|collection date:missing	Well P1 08B	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 B08\|rtindex:GCCATTCTCC\|genotype:UAS replicate2	GSM8775352	GSM8775352: Well P1 08B; Danio rerio; OTHER	GSM8775352 r1	GSM8775352	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-08B.R1.fastq.gz P1-08B.R2.fastq.gz P1-08B_S58_I1_001.fastq.gz	fastq fastq fastq	7182683524.0	78072647.0	GSM8775352 r1	0:34 1:48 2:10	A:1191235462;C:707748234;G:767219572;T:1075959763;N:5324025	34	48	10	1191235462	707748234	767219572	1075959763	5324025	SRX27573003	SRS23984657	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34734	34734	SRR32232946	SRX27573002	SRS23984655	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 08A	GSM8775351	source name:6 dpf head\|tissue:6 dpf head\|well:P1 A08\|rtindex:TGCGGACCTA\|genotype:UAS replicate2\|geo loc name:missing\|collection date:missing	Well P1 08A	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 A08\|rtindex:TGCGGACCTA\|genotype:UAS replicate2	GSM8775351	GSM8775351: Well P1 08A; Danio rerio; OTHER	GSM8775351 r1	GSM8775351	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-08A.R1.fastq.gz P1-08A.R2.fastq.gz P1-08A_S57_I1_001.fastq.gz	fastq fastq fastq	33057440.0	359320.0	GSM8775351 r1	0:34 1:48 2:10	A:5780741;C:2263389;G:5259112;T:3921095;N:23023	34	48	10	5780741	2263389	5259112	3921095	23023	SRX27573002	SRS23984655	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34735	34735	SRR32232947	SRX27573001	SRS23984654	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 04H	GSM8775326	source name:6 dpf head\|tissue:6 dpf head\|well:P1 H04\|rtindex:AGGTTAGCAT\|genotype:UAS replicate1\|geo loc name:missing\|collection date:missing	Well P1 04H	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 H04\|rtindex:AGGTTAGCAT\|genotype:UAS replicate1	GSM8775326	GSM8775326: Well P1 04H; Danio rerio; OTHER	GSM8775326 r1	GSM8775326	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-04H.R1.fastq.gz P1-04H.R2.fastq.gz P1-04H_S32_I1_001.fastq.gz	fastq fastq fastq	4048.0	44.0	GSM8775326 r1	0:34 1:48 2:10	A:877;C:264;G:476;T:495;N:0	34	48	10	877	264	476	495	0	SRX27573001	SRS23984654	SRA2070568	UMass Chan	UMass Chan	T	under 1.2% mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34736	34736	SRR32232948	SRX27573000	SRS23984653	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 04G	GSM8775325	source name:6 dpf head\|tissue:6 dpf head\|well:P1 G04\|rtindex:ACGAAGTCAA\|genotype:UAS replicate1\|geo loc name:missing\|collection date:missing	Well P1 04G	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 G04\|rtindex:ACGAAGTCAA\|genotype:UAS replicate1	GSM8775325	GSM8775325: Well P1 04G; Danio rerio; OTHER	GSM8775325 r1	GSM8775325	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-04G.R1.fastq.gz P1-04G.R2.fastq.gz P1-04G_S31_I1_001.fastq.gz	fastq fastq fastq	16717412.0	181711.0	GSM8775325 r1	0:34 1:48 2:10	A:2534077;C:1123957;G:3093004;T:1959215;N:11875	34	48	10	2534077	1123957	3093004	1959215	11875	SRX27573000	SRS23984653	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34737	34737	SRR32232949	SRX27572999	SRS23984652	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 04F	GSM8775324	source name:6 dpf head\|tissue:6 dpf head\|well:P1 F04\|rtindex:AATCGATAAT\|genotype:UAS replicate1\|geo loc name:missing\|collection date:missing	Well P1 04F	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 F04\|rtindex:AATCGATAAT\|genotype:UAS replicate1	GSM8775324	GSM8775324: Well P1 04F; Danio rerio; OTHER	GSM8775324 r1	GSM8775324	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-04F.R1.fastq.gz P1-04F.R2.fastq.gz P1-04F_S30_I1_001.fastq.gz	fastq fastq fastq	71229712.0	774236.0	GSM8775324 r1	0:34 1:48 2:10	A:13212435;C:4888655;G:10555214;T:8456988;N:50036	34	48	10	13212435	4888655	10555214	8456988	50036	SRX27572999	SRS23984652	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34738	34738	SRR32232950	SRX27572998	SRS23984651	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 04E	GSM8775323	source name:6 dpf head\|tissue:6 dpf head\|well:P1 E04\|rtindex:TAGCCGTCAT\|genotype:UAS replicate1\|geo loc name:missing\|collection date:missing	Well P1 04E	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 E04\|rtindex:TAGCCGTCAT\|genotype:UAS replicate1	GSM8775323	GSM8775323: Well P1 04E; Danio rerio; OTHER	GSM8775323 r1	GSM8775323	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-04E.R1.fastq.gz P1-04E.R2.fastq.gz P1-04E_S29_I1_001.fastq.gz	fastq fastq fastq	381955388.0	4151689.0	GSM8775323 r1	0:34 1:48 2:10	A:87514409;C:25262710;G:36461600;T:49768768;N:273585	34	48	10	87514409	25262710	36461600	49768768	273585	SRX27572998	SRS23984651	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34739	34739	SRR32232951	SRX27572997	SRS23984649	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 04D	GSM8775322	source name:6 dpf head\|tissue:6 dpf head\|well:P1 D04\|rtindex:TCCGGCCTCG\|genotype:UAS replicate1\|geo loc name:missing\|collection date:missing	Well P1 04D	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 D04\|rtindex:TCCGGCCTCG\|genotype:UAS replicate1	GSM8775322	GSM8775322: Well P1 04D; Danio rerio; OTHER	GSM8775322 r1	GSM8775322	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-04D.R1.fastq.gz P1-04D.R2.fastq.gz P1-04D_S28_I1_001.fastq.gz	fastq fastq fastq	57109552.0	620756.0	GSM8775322 r1	0:34 1:48 2:10	A:11045441;C:3910161;G:7896770;T:6905332;N:38584	34	48	10	11045441	3910161	7896770	6905332	38584	SRX27572997	SRS23984649	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34740	34740	SRR32232952	SRX27572996	SRS23984650	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 04C	GSM8775321	source name:6 dpf head\|tissue:6 dpf head\|well:P1 C04\|rtindex:TTATCCGGAT\|genotype:UAS replicate1\|geo loc name:missing\|collection date:missing	Well P1 04C	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 C04\|rtindex:TTATCCGGAT\|genotype:UAS replicate1	GSM8775321	GSM8775321: Well P1 04C; Danio rerio; OTHER	GSM8775321 r1	GSM8775321	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-04C.R1.fastq.gz P1-04C.R2.fastq.gz P1-04C_S27_I1_001.fastq.gz	fastq fastq fastq	41102932.0	446771.0	GSM8775321 r1	0:34 1:48 2:10	A:7556055;C:2874427;G:6121487;T:4864586;N:28453	34	48	10	7556055	2874427	6121487	4864586	28453	SRX27572996	SRS23984650	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34741	34741	SRR32232953	SRX27572995	SRS23984648	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 04B	GSM8775320	source name:6 dpf head\|tissue:6 dpf head\|well:P1 B04\|rtindex:ACTCAATAGG\|genotype:UAS replicate1\|geo loc name:missing\|collection date:missing	Well P1 04B	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 B04\|rtindex:ACTCAATAGG\|genotype:UAS replicate1	GSM8775320	GSM8775320: Well P1 04B; Danio rerio; OTHER	GSM8775320 r1	GSM8775320	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-04B.R1.fastq.gz P1-04B.R2.fastq.gz P1-04B_S26_I1_001.fastq.gz	fastq fastq fastq	39908772.0	433791.0	GSM8775320 r1	0:34 1:48 2:10	A:7097865;C:2701376;G:6191071;T:4805009;N:26647	34	48	10	7097865	2701376	6191071	4805009	26647	SRX27572995	SRS23984648	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34742	34742	SRR32232954	SRX27572994	SRS23984647	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 04A	GSM8775319	source name:6 dpf head\|tissue:6 dpf head\|well:P1 A04\|rtindex:CTGATGGTCA\|genotype:UAS replicate1\|geo loc name:missing\|collection date:missing	Well P1 04A	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 A04\|rtindex:CTGATGGTCA\|genotype:UAS replicate1	GSM8775319	GSM8775319: Well P1 04A; Danio rerio; OTHER	GSM8775319 r1	GSM8775319	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-04A.R1.fastq.gz P1-04A.R2.fastq.gz P1-04A_S25_I1_001.fastq.gz	fastq fastq fastq	74926548.0	814419.0	GSM8775319 r1	0:34 1:48 2:10	A:14546606;C:5316463;G:9739043;T:9434376;N:55624	34	48	10	14546606	5316463	9739043	9434376	55624	SRX27572994	SRS23984647	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34743	34743	SRR32232955	SRX27572993	SRS23984646	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 11A	GSM8775374	source name:6 dpf head\|tissue:6 dpf head\|well:P1 A11\|rtindex:AATTAAGACT\|genotype:GAL4UAS replicate3\|geo loc name:missing\|collection date:missing	Well P1 11A	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 A11\|rtindex:AATTAAGACT\|genotype:GAL4UAS replicate3	GSM8775374	GSM8775374: Well P1 11A; Danio rerio; OTHER	GSM8775374 r1	GSM8775374	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-11A.R1.fastq.gz P1-11A.R2.fastq.gz P1-11A_S81_I1_001.fastq.gz	fastq fastq fastq	23002760.0	250030.0	GSM8775374 r1	0:34 1:48 2:10	A:3455540;C:1563605;G:4264428;T:2701802;N:16065	34	48	10	3455540	1563605	4264428	2701802	16065	SRX27572993	SRS23984646	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34744	34744	SRR32232956	SRX27572992	SRS23984645	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 10G	GSM8775373	source name:6 dpf head\|tissue:6 dpf head\|well:P1 G10\|rtindex:ATCCATGACT\|genotype:wt replicate3\|geo loc name:missing\|collection date:missing	Well P1 10G	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 G10\|rtindex:ATCCATGACT\|genotype:wt replicate3	GSM8775373	GSM8775373: Well P1 10G; Danio rerio; OTHER	GSM8775373 r1	GSM8775373	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-10G.R1.fastq.gz P1-10G.R2.fastq.gz P1-10G_S79_I1_001.fastq.gz	fastq fastq fastq	7274440.0	79070.0	GSM8775373 r1	0:34 1:48 2:10	A:1025995;C:453768;G:1424925;T:885556;N:5116	34	48	10	1025995	453768	1424925	885556	5116	SRX27572992	SRS23984645	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34745	34745	SRR32232957	SRX27572991	SRS23984644	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 10F	GSM8775372	source name:6 dpf head\|tissue:6 dpf head\|well:P1 F10\|rtindex:GCTGGAACTT\|genotype:wt replicate3\|geo loc name:missing\|collection date:missing	Well P1 10F	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 F10\|rtindex:GCTGGAACTT\|genotype:wt replicate3	GSM8775372	GSM8775372: Well P1 10F; Danio rerio; OTHER	GSM8775372 r1	GSM8775372	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-10F.R1.fastq.gz P1-10F.R2.fastq.gz P1-10F_S78_I1_001.fastq.gz	fastq fastq fastq	32953020.0	358185.0	GSM8775372 r1	0:34 1:48 2:10	A:5148026;C:2283765;G:5798660;T:3938086;N:24343	34	48	10	5148026	2283765	5798660	3938086	24343	SRX27572991	SRS23984644	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34746	34746	SRR32232958	SRX27572990	SRS23984643	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 10E	GSM8775371	source name:6 dpf head\|tissue:6 dpf head\|well:P1 E10\|rtindex:TATATACTAA\|genotype:wt replicate3\|geo loc name:missing\|collection date:missing	Well P1 10E	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 E10\|rtindex:TATATACTAA\|genotype:wt replicate3	GSM8775371	GSM8775371: Well P1 10E; Danio rerio; OTHER	GSM8775371 r1	GSM8775371	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-10E.R1.fastq.gz P1-10E.R2.fastq.gz P1-10E_S77_I1_001.fastq.gz	fastq fastq fastq	7366056084.0	80065827.0	GSM8775371 r1	0:34 1:48 2:10	A:1137867832;C:775309617;G:881567529;T:1042828519;N:5586199	34	48	10	1137867832	775309617	881567529	1042828519	5586199	SRX27572990	SRS23984643	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34747	34747	SRR32232959	SRX27572989	SRS23984642	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 10D	GSM8775370	source name:6 dpf head\|tissue:6 dpf head\|well:P1 D10\|rtindex:ACCGCCAACC\|genotype:wt replicate3\|geo loc name:missing\|collection date:missing	Well P1 10D	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 D10\|rtindex:ACCGCCAACC\|genotype:wt replicate3	GSM8775370	GSM8775370: Well P1 10D; Danio rerio; OTHER	GSM8775370 r1	GSM8775370	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-10D.R1.fastq.gz P1-10D.R2.fastq.gz P1-10D_S76_I1_001.fastq.gz	fastq fastq fastq	32482532.0	353071.0	GSM8775370 r1	0:34 1:48 2:10	A:4191254;C:2035744;G:6991172;T:3707782;N:21456	34	48	10	4191254	2035744	6991172	3707782	21456	SRX27572989	SRS23984642	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34748	34748	SRR32232960	SRX27572988	SRS23984641	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 10C	GSM8775369	source name:6 dpf head\|tissue:6 dpf head\|well:P1 C10\|rtindex:ACTTAACCTT\|genotype:wt replicate3\|geo loc name:missing\|collection date:missing	Well P1 10C	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 C10\|rtindex:ACTTAACCTT\|genotype:wt replicate3	GSM8775369	GSM8775369: Well P1 10C; Danio rerio; OTHER	GSM8775369 r1	GSM8775369	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-10C.R1.fastq.gz P1-10C.R2.fastq.gz P1-10C_S75_I1_001.fastq.gz	fastq fastq fastq	29742312.0	323286.0	GSM8775369 r1	0:34 1:48 2:10	A:4641549;C:2072399;G:5265185;T:3517381;N:21214	34	48	10	4641549	2072399	5265185	3517381	21214	SRX27572988	SRS23984641	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34749	34749	SRR32232961	SRX27572987	SRS23984640	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 10B	GSM8775368	source name:6 dpf head\|tissue:6 dpf head\|well:P1 B10\|rtindex:TTACCGAGGC\|genotype:wt replicate3\|geo loc name:missing\|collection date:missing	Well P1 10B	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 B10\|rtindex:TTACCGAGGC\|genotype:wt replicate3	GSM8775368	GSM8775368: Well P1 10B; Danio rerio; OTHER	GSM8775368 r1	GSM8775368	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-10B.R1.fastq.gz P1-10B.R2.fastq.gz P1-10B_S74_I1_001.fastq.gz	fastq fastq fastq	23657432.0	257146.0	GSM8775368 r1	0:34 1:48 2:10	A:3666551;C:1613324;G:4291430;T:2755799;N:15904	34	48	10	3666551	1613324	4291430	2755799	15904	SRX27572987	SRS23984640	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34750	34750	SRR32232962	SRX27572986	SRS23984639	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 10A	GSM8775367	source name:6 dpf head\|tissue:6 dpf head\|well:P1 A10\|rtindex:TAGATCTACT\|genotype:wt replicate3\|geo loc name:missing\|collection date:missing	Well P1 10A	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 A10\|rtindex:TAGATCTACT\|genotype:wt replicate3	GSM8775367	GSM8775367: Well P1 10A; Danio rerio; OTHER	GSM8775367 r1	GSM8775367	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-10A.R1.fastq.gz P1-10A.R2.fastq.gz P1-10A_S73_I1_001.fastq.gz	fastq fastq fastq	20690064.0	224892.0	GSM8775367 r1	0:34 1:48 2:10	A:3162846;C:1394801;G:3805546;T:2416575;N:15048	34	48	10	3162846	1394801	3805546	2416575	15048	SRX27572986	SRS23984639	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34751	34751	SRR32232963	SRX27572985	SRS23984638	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 06H	GSM8775342	source name:6 dpf head\|tissue:6 dpf head\|well:P1 H06\|rtindex:AGAGCCAAGG\|genotype:wt replicate2\|geo loc name:missing\|collection date:missing	Well P1 06H	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 H06\|rtindex:AGAGCCAAGG\|genotype:wt replicate2	GSM8775342	GSM8775342: Well P1 06H; Danio rerio; OTHER	GSM8775342 r1	GSM8775342	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-06H.R1.fastq.gz P1-06H.R2.fastq.gz P1-06H_S48_I1_001.fastq.gz	fastq fastq fastq	63664.0	692.0	GSM8775342 r1	0:34 1:48 2:10	A:13330;C:4382;G:7530;T:7889;N:85	34	48	10	13330	4382	7530	7889	85	SRX27572985	SRS23984638	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34752	34752	SRR32232964	SRX27572984	SRS23984637	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 06G	GSM8775341	source name:6 dpf head\|tissue:6 dpf head\|well:P1 G06\|rtindex:GGAGGATAGC\|genotype:wt replicate2\|geo loc name:missing\|collection date:missing	Well P1 06G	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 G06\|rtindex:GGAGGATAGC\|genotype:wt replicate2	GSM8775341	GSM8775341: Well P1 06G; Danio rerio; OTHER	GSM8775341 r1	GSM8775341	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-06G.R1.fastq.gz P1-06G.R2.fastq.gz P1-06G_S47_I1_001.fastq.gz	fastq fastq fastq	35465816.0	385498.0	GSM8775341 r1	0:34 1:48 2:10	A:5266761;C:2313383;G:6547005;T:4353297;N:23458	34	48	10	5266761	2313383	6547005	4353297	23458	SRX27572984	SRS23984637	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34753	34753	SRR32232965	SRX27572983	SRS23984636	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 06F	GSM8775340	source name:6 dpf head\|tissue:6 dpf head\|well:P1 F06\|rtindex:TCAACGTAAG\|genotype:wt replicate2\|geo loc name:missing\|collection date:missing	Well P1 06F	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 F06\|rtindex:TCAACGTAAG\|genotype:wt replicate2	GSM8775340	GSM8775340: Well P1 06F; Danio rerio; OTHER	GSM8775340 r1	GSM8775340	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-06F.R1.fastq.gz P1-06F.R2.fastq.gz P1-06F_S46_I1_001.fastq.gz	fastq fastq fastq	40023036.0	435033.0	GSM8775340 r1	0:34 1:48 2:10	A:6489618;C:2824422;G:6833067;T:4706650;N:27827	34	48	10	6489618	2824422	6833067	4706650	27827	SRX27572983	SRS23984636	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34754	34754	SRR32232966	SRX27572982	SRS23984635	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 06E	GSM8775339	source name:6 dpf head\|tissue:6 dpf head\|well:P1 E06\|rtindex:ACGCGAGATT\|genotype:wt replicate2\|geo loc name:missing\|collection date:missing	Well P1 06E	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 E06\|rtindex:ACGCGAGATT\|genotype:wt replicate2	GSM8775339	GSM8775339: Well P1 06E; Danio rerio; OTHER	GSM8775339 r1	GSM8775339	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-06E.R1.fastq.gz P1-06E.R2.fastq.gz P1-06E_S45_I1_001.fastq.gz	fastq fastq fastq	59610020.0	647935.0	GSM8775339 r1	0:34 1:48 2:10	A:10804415;C:4165808;G:9006405;T:7083045;N:41207	34	48	10	10804415	4165808	9006405	7083045	41207	SRX27572982	SRS23984635	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34755	34755	SRR32232967	SRX27572981	SRS23984633	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 06D	GSM8775338	source name:6 dpf head\|tissue:6 dpf head\|well:P1 D06\|rtindex:CATACTCCGA\|genotype:wt replicate2\|geo loc name:missing\|collection date:missing	Well P1 06D	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 D06\|rtindex:CATACTCCGA\|genotype:wt replicate2	GSM8775338	GSM8775338: Well P1 06D; Danio rerio; OTHER	GSM8775338 r1	GSM8775338	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-06D.R1.fastq.gz P1-06D.R2.fastq.gz P1-06D_S44_I1_001.fastq.gz	fastq fastq fastq	4944350388.0	53742939.0	GSM8775338 r1	0:34 1:48 2:10	A:924216784;C:417859577;G:514301359;T:719732920;N:3550432	34	48	10	924216784	417859577	514301359	719732920	3550432	SRX27572981	SRS23984633	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34756	34756	SRR32232968	SRX27572980	SRS23984634	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 06C	GSM8775337	source name:6 dpf head\|tissue:6 dpf head\|well:P1 C06\|rtindex:GCAGGTCCGT\|genotype:wt replicate2\|geo loc name:missing\|collection date:missing	Well P1 06C	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 C06\|rtindex:GCAGGTCCGT\|genotype:wt replicate2	GSM8775337	GSM8775337: Well P1 06C; Danio rerio; OTHER	GSM8775337 r1	GSM8775337	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-06C.R1.fastq.gz P1-06C.R2.fastq.gz P1-06C_S43_I1_001.fastq.gz	fastq fastq fastq	37857448.0	411494.0	GSM8775337 r1	0:34 1:48 2:10	A:6636308;C:2636497;G:5984141;T:4469989;N:24777	34	48	10	6636308	2636497	5984141	4469989	24777	SRX27572980	SRS23984634	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34757	34757	SRR32232969	SRX27572979	SRS23984632	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 06B	GSM8775336	source name:6 dpf head\|tissue:6 dpf head\|well:P1 B06\|rtindex:GGTACTGCCT\|genotype:wt replicate2\|geo loc name:missing\|collection date:missing	Well P1 06B	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 B06\|rtindex:GGTACTGCCT\|genotype:wt replicate2	GSM8775336	GSM8775336: Well P1 06B; Danio rerio; OTHER	GSM8775336 r1	GSM8775336	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-06B.R1.fastq.gz P1-06B.R2.fastq.gz P1-06B_S42_I1_001.fastq.gz	fastq fastq fastq	36989796.0	402063.0	GSM8775336 r1	0:34 1:48 2:10	A:6103502;C:2581732;G:6111203;T:4475906;N:26681	34	48	10	6103502	2581732	6111203	4475906	26681	SRX27572979	SRS23984632	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34758	34758	SRR32232970	SRX27572978	SRS23984631	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 06A	GSM8775335	source name:6 dpf head\|tissue:6 dpf head\|well:P1 A06\|rtindex:GCCGCAACGA\|genotype:wt replicate2\|geo loc name:missing\|collection date:missing	Well P1 06A	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 A06\|rtindex:GCCGCAACGA\|genotype:wt replicate2	GSM8775335	GSM8775335: Well P1 06A; Danio rerio; OTHER	GSM8775335 r1	GSM8775335	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-06A.R1.fastq.gz P1-06A.R2.fastq.gz P1-06A_S41_I1_001.fastq.gz	fastq fastq fastq	181733028.0	1975359.0	GSM8775335 r1	0:34 1:48 2:10	A:39079683;C:12211237;G:19042922;T:24349968;N:133422	34	48	10	39079683	12211237	19042922	24349968	133422	SRX27572978	SRS23984631	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34759	34759	SRR32232971	SRX27572977	SRS23984630	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 02H	GSM8775310	source name:6 dpf head\|tissue:6 dpf head\|well:P1 H02\|rtindex:TCTTGCCGAC\|genotype:wt replicate1\|geo loc name:missing\|collection date:missing	Well P1 02H	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 H02\|rtindex:TCTTGCCGAC\|genotype:wt replicate1	GSM8775310	GSM8775310: Well P1 02H; Danio rerio; OTHER	GSM8775310 r1	GSM8775310	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-02H.R1.fastq.gz P1-02H.R2.fastq.gz P1-02H_S16_I1_001.fastq.gz	fastq fastq fastq	460.0	5.0	GSM8775310 r1	0:34 1:48 2:10	A:79;C:22;G:97;T:42;N:0	34	48	10	79	22	97	42	0	SRX27572977	SRS23984630	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34760	34760	SRR32232972	SRX27572976	SRS23984629	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 02G	GSM8775309	source name:6 dpf head\|tissue:6 dpf head\|well:P1 G02\|rtindex:CGGATTAGAA\|genotype:wt replicate1\|geo loc name:missing\|collection date:missing	Well P1 02G	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 G02\|rtindex:CGGATTAGAA\|genotype:wt replicate1	GSM8775309	GSM8775309: Well P1 02G; Danio rerio; OTHER	GSM8775309 r1	GSM8775309	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-02G.R1.fastq.gz P1-02G.R2.fastq.gz P1-02G_S15_I1_001.fastq.gz	fastq fastq fastq	100724268.0	1094829.0	GSM8775309 r1	0:34 1:48 2:10	A:20865846;C:7071976;G:10244265;T:14295638;N:74067	34	48	10	20865846	7071976	10244265	14295638	74067	SRX27572976	SRS23984629	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34761	34761	SRR32232973	SRX27572975	SRS23984628	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 02F	GSM8775308	source name:6 dpf head\|tissue:6 dpf head\|well:P1 F02\|rtindex:CGTAGTTACC\|genotype:wt replicate1\|geo loc name:missing\|collection date:missing	Well P1 02F	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 F02\|rtindex:CGTAGTTACC\|genotype:wt replicate1	GSM8775308	GSM8775308: Well P1 02F; Danio rerio; OTHER	GSM8775308 r1	GSM8775308	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-02F.R1.fastq.gz P1-02F.R2.fastq.gz P1-02F_S14_I1_001.fastq.gz	fastq fastq fastq	397078348.0	4316069.0	GSM8775308 r1	0:34 1:48 2:10	A:92040782;C:26097382;G:36181559;T:52564018;N:287571	34	48	10	92040782	26097382	36181559	52564018	287571	SRX27572975	SRS23984628	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34762	34762	SRR32232974	SRX27572974	SRS23984627	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 02E	GSM8775307	source name:6 dpf head\|tissue:6 dpf head\|well:P1 E02\|rtindex:AACCTGCGTA\|genotype:wt replicate1\|geo loc name:missing\|collection date:missing	Well P1 02E	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 E02\|rtindex:AACCTGCGTA\|genotype:wt replicate1	GSM8775307	GSM8775307: Well P1 02E; Danio rerio; OTHER	GSM8775307 r1	GSM8775307	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-02E.R1.fastq.gz P1-02E.R2.fastq.gz P1-02E_S13_I1_001.fastq.gz	fastq fastq fastq	97598568.0	1060854.0	GSM8775307 r1	0:34 1:48 2:10	A:20986490;C:6417842;G:11267196;T:12179365;N:70099	34	48	10	20986490	6417842	11267196	12179365	70099	SRX27572974	SRS23984627	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34763	34763	SRR32232975	SRX27572973	SRS23984625	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 02D	GSM8775306	source name:6 dpf head\|tissue:6 dpf head\|well:P1 D02\|rtindex:GAAGATCGAG\|genotype:wt replicate1\|geo loc name:missing\|collection date:missing	Well P1 02D	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 D02\|rtindex:GAAGATCGAG\|genotype:wt replicate1	GSM8775306	GSM8775306: Well P1 02D; Danio rerio; OTHER	GSM8775306 r1	GSM8775306	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-02D.R1.fastq.gz P1-02D.R2.fastq.gz P1-02D_S12_I1_001.fastq.gz	fastq fastq fastq	4315783480.0	46910690.0	GSM8775306 r1	0:34 1:48 2:10	A:851820437;C:342635980;G:430524180;T:623563815;N:3168708	34	48	10	851820437	342635980	430524180	623563815	3168708	SRX27572973	SRS23984625	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34764	34764	SRR32232976	SRX27572972	SRS23984626	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 02C	GSM8775305	source name:6 dpf head\|tissue:6 dpf head\|well:P1 C02\|rtindex:CGACGCGACT\|genotype:wt replicate1\|geo loc name:missing\|collection date:missing	Well P1 02C	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 C02\|rtindex:CGACGCGACT\|genotype:wt replicate1	GSM8775305	GSM8775305: Well P1 02C; Danio rerio; OTHER	GSM8775305 r1	GSM8775305	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-02C.R1.fastq.gz P1-02C.R2.fastq.gz P1-02C_S11_I1_001.fastq.gz	fastq fastq fastq	62879608.0	683474.0	GSM8775305 r1	0:34 1:48 2:10	A:12210765;C:4319771;G:8466498;T:7766050;N:43668	34	48	10	12210765	4319771	8466498	7766050	43668	SRX27572972	SRS23984626	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34765	34765	SRR32232977	SRX27572971	SRS23984623	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 02B	GSM8775304	source name:6 dpf head\|tissue:6 dpf head\|well:P1 B02\|rtindex:AGAGCTATAA\|genotype:wt replicate1\|geo loc name:missing\|collection date:missing	Well P1 02B	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 B02\|rtindex:AGAGCTATAA\|genotype:wt replicate1	GSM8775304	GSM8775304: Well P1 02B; Danio rerio; OTHER	GSM8775304 r1	GSM8775304	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-02B.R1.fastq.gz P1-02B.R2.fastq.gz P1-02B_S10_I1_001.fastq.gz	fastq fastq fastq	43401920.0	471760.0	GSM8775304 r1	0:34 1:48 2:10	A:8440271;C:2935979;G:6044455;T:5195314;N:28461	34	48	10	8440271	2935979	6044455	5195314	28461	SRX27572971	SRS23984623	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34766	34766	SRR32232978	SRX27572970	SRS23984622	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 02A	GSM8775303	source name:6 dpf head\|tissue:6 dpf head\|well:P1 A02\|rtindex:GCGTTGGAGC\|genotype:wt replicate1\|geo loc name:missing\|collection date:missing	Well P1 02A	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 A02\|rtindex:GCGTTGGAGC\|genotype:wt replicate1	GSM8775303	GSM8775303: Well P1 02A; Danio rerio; OTHER	GSM8775303 r1	GSM8775303	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-02A.R1.fastq.gz P1-02A.R2.fastq.gz P1-02A_S9_I1_001.fastq.gz	fastq fastq fastq	461011540.0	5010995.0	GSM8775303 r1	0:34 1:48 2:10	A:104776330;C:30925787;G:42506931;T:61984548;N:334164	34	48	10	104776330	30925787	42506931	61984548	334164	SRX27572970	SRS23984622	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34767	34767	SRR32232979	SRX27572969	SRS23984624	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 12A	GSM8775382	source name:6 dpf head\|tissue:6 dpf head\|well:P1 A12\|rtindex:TTATTCATTC\|genotype:UAS replicate3\|geo loc name:missing\|collection date:missing	Well P1 12A	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 A12\|rtindex:TTATTCATTC\|genotype:UAS replicate3	GSM8775382	GSM8775382: Well P1 12A; Danio rerio; OTHER	GSM8775382 r1	GSM8775382	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-12A.R1.fastq.gz P1-12A.R2.fastq.gz P1-12A_S89_I1_001.fastq.gz	fastq fastq fastq	16741424.0	181972.0	GSM8775382 r1	0:34 1:48 2:10	A:2677910;C:1120864;G:2940806;T:1984541;N:10535	34	48	10	2677910	1120864	2940806	1984541	10535	SRX27572969	SRS23984624	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34768	34768	SRR32232980	SRX27572968	SRS23984621	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 11H	GSM8775381	source name:6 dpf head\|tissue:6 dpf head\|well:P1 H11\|rtindex:ACCTGGCCAA\|genotype:GAL4UAS replicate3\|geo loc name:missing\|collection date:missing	Well P1 11H	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 H11\|rtindex:ACCTGGCCAA\|genotype:GAL4UAS replicate3	GSM8775381	GSM8775381: Well P1 11H; Danio rerio; OTHER	GSM8775381 r1	GSM8775381	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-11H.R1.fastq.gz P1-11H.R2.fastq.gz P1-11H_S88_I1_001.fastq.gz	fastq fastq fastq	920.0	10.0	GSM8775381 r1	0:34 1:48 2:10	A:198;C:61;G:86;T:135;N:0	34	48	10	198	61	86	135	0	SRX27572968	SRS23984621	SRA2070568	UMass Chan	UMass Chan	T	under 1.2% mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34769	34769	SRR32232981	SRX27572967	SRS23984620	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 11G	GSM8775380	source name:6 dpf head\|tissue:6 dpf head\|well:P1 G11\|rtindex:AACCTGGTCT\|genotype:GAL4UAS replicate3\|geo loc name:missing\|collection date:missing	Well P1 11G	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 G11\|rtindex:AACCTGGTCT\|genotype:GAL4UAS replicate3	GSM8775380	GSM8775380: Well P1 11G; Danio rerio; OTHER	GSM8775380 r1	GSM8775380	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-11G.R1.fastq.gz P1-11G.R2.fastq.gz P1-11G_S87_I1_001.fastq.gz	fastq fastq fastq	47417168.0	515404.0	GSM8775380 r1	0:34 1:48 2:10	A:6801318;C:3146390;G:8860580;T:5896979;N:34125	34	48	10	6801318	3146390	8860580	5896979	34125	SRX27572967	SRS23984620	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34770	34770	SRR32232982	SRX27572966	SRS23984619	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 11F	GSM8775379	source name:6 dpf head\|tissue:6 dpf head\|well:P1 F11\|rtindex:AGGTTAGTTC\|genotype:GAL4UAS replicate3\|geo loc name:missing\|collection date:missing	Well P1 11F	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 F11\|rtindex:AGGTTAGTTC\|genotype:GAL4UAS replicate3	GSM8775379	GSM8775379: Well P1 11F; Danio rerio; OTHER	GSM8775379 r1	GSM8775379	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-11F.R1.fastq.gz P1-11F.R2.fastq.gz P1-11F_S86_I1_001.fastq.gz	fastq fastq fastq	31114768.0	338204.0	GSM8775379 r1	0:34 1:48 2:10	A:4790024;C:2139061;G:5605750;T:3678276;N:20681	34	48	10	4790024	2139061	5605750	3678276	20681	SRX27572966	SRS23984619	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34771	34771	SRR32232983	SRX27572965	SRS23984618	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 11E	GSM8775378	source name:6 dpf head\|tissue:6 dpf head\|well:P1 E11\|rtindex:ACTTGCTAGA\|genotype:GAL4UAS replicate3\|geo loc name:missing\|collection date:missing	Well P1 11E	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 E11\|rtindex:ACTTGCTAGA\|genotype:GAL4UAS replicate3	GSM8775378	GSM8775378: Well P1 11E; Danio rerio; OTHER	GSM8775378 r1	GSM8775378	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-11E.R1.fastq.gz P1-11E.R2.fastq.gz P1-11E_S85_I1_001.fastq.gz	fastq fastq fastq	30435440.0	330820.0	GSM8775378 r1	0:34 1:48 2:10	A:4748511;C:2122090;G:5400332;T:3588118;N:20309	34	48	10	4748511	2122090	5400332	3588118	20309	SRX27572965	SRS23984618	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34772	34772	SRR32232984	SRX27572964	SRS23984617	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 11D	GSM8775377	source name:6 dpf head\|tissue:6 dpf head\|well:P1 D11\|rtindex:ATAAGGAGCA\|genotype:GAL4UAS replicate3\|geo loc name:missing\|collection date:missing	Well P1 11D	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 D11\|rtindex:ATAAGGAGCA\|genotype:GAL4UAS replicate3	GSM8775377	GSM8775377: Well P1 11D; Danio rerio; OTHER	GSM8775377 r1	GSM8775377	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-11D.R1.fastq.gz P1-11D.R2.fastq.gz P1-11D_S84_I1_001.fastq.gz	fastq fastq fastq	28003972.0	304391.0	GSM8775377 r1	0:34 1:48 2:10	A:4493127;C:1907680;G:4963314;T:3228507;N:18140	34	48	10	4493127	1907680	4963314	3228507	18140	SRX27572964	SRS23984617	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34773	34773	SRR32232985	SRX27572963	SRS23984616	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 11C	GSM8775376	source name:6 dpf head\|tissue:6 dpf head\|well:P1 C11\|rtindex:CAACCGCTAA\|genotype:GAL4UAS replicate3\|geo loc name:missing\|collection date:missing	Well P1 11C	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 C11\|rtindex:CAACCGCTAA\|genotype:GAL4UAS replicate3	GSM8775376	GSM8775376: Well P1 11C; Danio rerio; OTHER	GSM8775376 r1	GSM8775376	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-11C.R1.fastq.gz P1-11C.R2.fastq.gz P1-11C_S83_I1_001.fastq.gz	fastq fastq fastq	21477952.0	233456.0	GSM8775376 r1	0:34 1:48 2:10	A:3270849;C:1474502;G:3915416;T:2530065;N:15056	34	48	10	3270849	1474502	3915416	2530065	15056	SRX27572963	SRS23984616	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34774	34774	SRR32232986	SRX27572962	SRS23984615	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 11B	GSM8775375	source name:6 dpf head\|tissue:6 dpf head\|well:P1 B11\|rtindex:ATCATATTAG\|genotype:GAL4UAS replicate3\|geo loc name:missing\|collection date:missing	Well P1 11B	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 B11\|rtindex:ATCATATTAG\|genotype:GAL4UAS replicate3	GSM8775375	GSM8775375: Well P1 11B; Danio rerio; OTHER	GSM8775375 r1	GSM8775375	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-11B.R1.fastq.gz P1-11B.R2.fastq.gz P1-11B_S82_I1_001.fastq.gz	fastq fastq fastq	21279600.0	231300.0	GSM8775375 r1	0:34 1:48 2:10	A:3177194;C:1463431;G:3934752;T:2512075;N:14948	34	48	10	3177194	1463431	3934752	2512075	14948	SRX27572962	SRS23984615	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34775	34775	SRR32232987	SRX27572961	SRS23984614	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 07H	GSM8775350	source name:6 dpf head\|tissue:6 dpf head\|well:P1 H07\|rtindex:AATACCATCC\|genotype:GAL4UAS replicate2\|geo loc name:missing\|collection date:missing	Well P1 07H	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 H07\|rtindex:AATACCATCC\|genotype:GAL4UAS replicate2	GSM8775350	GSM8775350: Well P1 07H; Danio rerio; OTHER	GSM8775350 r1	GSM8775350	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-07H.R1.fastq.gz P1-07H.R2.fastq.gz P1-07H_S56_I1_001.fastq.gz	fastq fastq fastq	644.0	7.0	GSM8775350 r1	0:34 1:48 2:10	A:110;C:48;G:94;T:84;N:0	34	48	10	110	48	94	84	0	SRX27572961	SRS23984614	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34776	34776	SRR32232988	SRX27572960	SRS23984612	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 07G	GSM8775349	source name:6 dpf head\|tissue:6 dpf head\|well:P1 G07\|rtindex:GGCTCTCTAT\|genotype:GAL4UAS replicate2\|geo loc name:missing\|collection date:missing	Well P1 07G	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 G07\|rtindex:GGCTCTCTAT\|genotype:GAL4UAS replicate2	GSM8775349	GSM8775349: Well P1 07G; Danio rerio; OTHER	GSM8775349 r1	GSM8775349	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-07G.R1.fastq.gz P1-07G.R2.fastq.gz P1-07G_S55_I1_001.fastq.gz	fastq fastq fastq	837346556.0	9101593.0	GSM8775349 r1	0:34 1:48 2:10	A:181851041;C:59310804;G:74090148;T:121010089;N:614382	34	48	10	181851041	59310804	74090148	121010089	614382	SRX27572960	SRS23984612	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34777	34777	SRR32232989	SRX27572959	SRS23984613	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 07F	GSM8775348	source name:6 dpf head\|tissue:6 dpf head\|well:P1 F07\|rtindex:TCTAATAGTA\|genotype:GAL4UAS replicate2\|geo loc name:missing\|collection date:missing	Well P1 07F	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 F07\|rtindex:TCTAATAGTA\|genotype:GAL4UAS replicate2	GSM8775348	GSM8775348: Well P1 07F; Danio rerio; OTHER	GSM8775348 r1	GSM8775348	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-07F.R1.fastq.gz P1-07F.R2.fastq.gz P1-07F_S54_I1_001.fastq.gz	fastq fastq fastq	51998308.0	565199.0	GSM8775348 r1	0:34 1:48 2:10	A:8691899;C:3542473;G:8590067;T:6268755;N:36358	34	48	10	8691899	3542473	8590067	6268755	36358	SRX27572959	SRS23984613	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34778	34778	SRR32232990	SRX27572958	SRS23984611	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 07E	GSM8775347	source name:6 dpf head\|tissue:6 dpf head\|well:P1 E07\|rtindex:GGTATCCGCC\|genotype:GAL4UAS replicate2\|geo loc name:missing\|collection date:missing	Well P1 07E	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 E07\|rtindex:GGTATCCGCC\|genotype:GAL4UAS replicate2	GSM8775347	GSM8775347: Well P1 07E; Danio rerio; OTHER	GSM8775347 r1	GSM8775347	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-07E.R1.fastq.gz P1-07E.R2.fastq.gz P1-07E_S53_I1_001.fastq.gz	fastq fastq fastq	4757958664.0	51716942.0	GSM8775347 r1	0:34 1:48 2:10	A:917767286;C:388310145;G:453981093;T:718871529;N:3483163	34	48	10	917767286	388310145	453981093	718871529	3483163	SRX27572958	SRS23984611	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34779	34779	SRR32232991	SRX27572957	SRS23984610	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 07D	GSM8775346	source name:6 dpf head\|tissue:6 dpf head\|well:P1 D07\|rtindex:GCTAACTTGC\|genotype:GAL4UAS replicate2\|geo loc name:missing\|collection date:missing	Well P1 07D	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 D07\|rtindex:GCTAACTTGC\|genotype:GAL4UAS replicate2	GSM8775346	GSM8775346: Well P1 07D; Danio rerio; OTHER	GSM8775346 r1	GSM8775346	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-07D.R1.fastq.gz P1-07D.R2.fastq.gz P1-07D_S52_I1_001.fastq.gz	fastq fastq fastq	33692056.0	366218.0	GSM8775346 r1	0:34 1:48 2:10	A:5438617;C:2339082;G:5799183;T:3978938;N:22644	34	48	10	5438617	2339082	5799183	3978938	22644	SRX27572957	SRS23984610	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34780	34780	SRR32232992	SRX27572956	SRS23984609	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 07C	GSM8775345	source name:6 dpf head\|tissue:6 dpf head\|well:P1 C07\|rtindex:TCGGCCTTAC\|genotype:GAL4UAS replicate2\|geo loc name:missing\|collection date:missing	Well P1 07C	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 C07\|rtindex:TCGGCCTTAC\|genotype:GAL4UAS replicate2	GSM8775345	GSM8775345: Well P1 07C; Danio rerio; OTHER	GSM8775345 r1	GSM8775345	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-07C.R1.fastq.gz P1-07C.R2.fastq.gz P1-07C_S51_I1_001.fastq.gz	fastq fastq fastq	1594757348.0	17334319.0	GSM8775345 r1	0:34 1:48 2:10	A:266696003;C:139558591;G:158516635;T:266107758;N:1168325	34	48	10	266696003	139558591	158516635	266107758	1168325	SRX27572956	SRS23984609	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34781	34781	SRR32232993	SRX27572955	SRS23984608	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 07B	GSM8775344	source name:6 dpf head\|tissue:6 dpf head\|well:P1 B07\|rtindex:TAGAATTAAC\|genotype:GAL4UAS replicate2\|geo loc name:missing\|collection date:missing	Well P1 07B	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 B07\|rtindex:TAGAATTAAC\|genotype:GAL4UAS replicate2	GSM8775344	GSM8775344: Well P1 07B; Danio rerio; OTHER	GSM8775344 r1	GSM8775344	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-07B.R1.fastq.gz P1-07B.R2.fastq.gz P1-07B_S50_I1_001.fastq.gz	fastq fastq fastq	30341600.0	329800.0	GSM8775344 r1	0:34 1:48 2:10	A:4935051;C:2157330;G:5157156;T:3559714;N:21149	34	48	10	4935051	2157330	5157156	3559714	21149	SRX27572955	SRS23984608	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34782	34782	SRR32232994	SRX27572954	SRS23984607	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 07A	GSM8775343	source name:6 dpf head\|tissue:6 dpf head\|well:P1 A07\|rtindex:TGAGTCTGGC\|genotype:GAL4UAS replicate2\|geo loc name:missing\|collection date:missing	Well P1 07A	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 A07\|rtindex:TGAGTCTGGC\|genotype:GAL4UAS replicate2	GSM8775343	GSM8775343: Well P1 07A; Danio rerio; OTHER	GSM8775343 r1	GSM8775343	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-07A.R1.fastq.gz P1-07A.R2.fastq.gz P1-07A_S49_I1_001.fastq.gz	fastq fastq fastq	51962060.0	564805.0	GSM8775343 r1	0:34 1:48 2:10	A:9098744;C:3684168;G:8377271;T:5913518;N:36939	34	48	10	9098744	3684168	8377271	5913518	36939	SRX27572954	SRS23984607	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34783	34783	SRR32232995	SRX27572953	SRS23984606	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 03H	GSM8775318	source name:6 dpf head\|tissue:6 dpf head\|well:P1 H03\|rtindex:AAGACCGTTG\|genotype:GAL4UAS replicate1\|geo loc name:missing\|collection date:missing	Well P1 03H	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 H03\|rtindex:AAGACCGTTG\|genotype:GAL4UAS replicate1	GSM8775318	GSM8775318: Well P1 03H; Danio rerio; OTHER	GSM8775318 r1	GSM8775318	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-03H.R1.fastq.gz P1-03H.R2.fastq.gz P1-03H_S24_I1_001.fastq.gz	fastq fastq fastq	1012.0	11.0	GSM8775318 r1	0:34 1:48 2:10	A:194;C:60;G:140;T:134;N:0	34	48	10	194	60	140	134	0	SRX27572953	SRS23984606	SRA2070568	UMass Chan	UMass Chan			illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34784	34784	SRR32232996	SRX27572952	SRS23984605	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 03G	GSM8775317	source name:6 dpf head\|tissue:6 dpf head\|well:P1 G03\|rtindex:ATCGGCTATC\|genotype:GAL4UAS replicate1\|geo loc name:missing\|collection date:missing	Well P1 03G	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 G03\|rtindex:ATCGGCTATC\|genotype:GAL4UAS replicate1	GSM8775317	GSM8775317: Well P1 03G; Danio rerio; OTHER	GSM8775317 r1	GSM8775317	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-03G.R1.fastq.gz P1-03G.R2.fastq.gz P1-03G_S23_I1_001.fastq.gz	fastq fastq fastq	10973024.0	119272.0	GSM8775317 r1	0:34 1:48 2:10	A:1618652;C:710901;G:2106027;T:1282180;N:7296	34	48	10	1618652	710901	2106027	1282180	7296	SRX27572952	SRS23984605	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34785	34785	SRR32232997	SRX27572951	SRS23984604	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 03F	GSM8775316	source name:6 dpf head\|tissue:6 dpf head\|well:P1 F03\|rtindex:TCCAATCATC\|genotype:GAL4UAS replicate1\|geo loc name:missing\|collection date:missing	Well P1 03F	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 F03\|rtindex:TCCAATCATC\|genotype:GAL4UAS replicate1	GSM8775316	GSM8775316: Well P1 03F; Danio rerio; OTHER	GSM8775316 r1	GSM8775316	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-03F.R1.fastq.gz P1-03F.R2.fastq.gz P1-03F_S22_I1_001.fastq.gz	fastq fastq fastq	143677228.0	1561709.0	GSM8775316 r1	0:34 1:48 2:10	A:32106494;C:9327944;G:15133487;T:18290237;N:103870	34	48	10	32106494	9327944	15133487	18290237	103870	SRX27572951	SRS23984604	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34786	34786	SRR32232998	SRX27572950	SRS23984603	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 03E	GSM8775315	source name:6 dpf head\|tissue:6 dpf head\|well:P1 E03\|rtindex:GGCAGACGCC\|genotype:GAL4UAS replicate1\|geo loc name:missing\|collection date:missing	Well P1 03E	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 E03\|rtindex:GGCAGACGCC\|genotype:GAL4UAS replicate1	GSM8775315	GSM8775315: Well P1 03E; Danio rerio; OTHER	GSM8775315 r1	GSM8775315	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-03E.R1.fastq.gz P1-03E.R2.fastq.gz P1-03E_S21_I1_001.fastq.gz	fastq fastq fastq	140146084.0	1523327.0	GSM8775315 r1	0:34 1:48 2:10	A:31463127;C:9060717;G:14802782;T:17693205;N:99865	34	48	10	31463127	9060717	14802782	17693205	99865	SRX27572950	SRS23984603	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34787	34787	SRR32232999	SRX27572949	SRS23984602	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 03D	GSM8775314	source name:6 dpf head\|tissue:6 dpf head\|well:P1 D03\|rtindex:AAGAAGCTAG\|genotype:GAL4UAS replicate1\|geo loc name:missing\|collection date:missing	Well P1 03D	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 D03\|rtindex:AAGAAGCTAG\|genotype:GAL4UAS replicate1	GSM8775314	GSM8775314: Well P1 03D; Danio rerio; OTHER	GSM8775314 r1	GSM8775314	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-03D.R1.fastq.gz P1-03D.R2.fastq.gz P1-03D_S20_I1_001.fastq.gz	fastq fastq fastq	37007184.0	402252.0	GSM8775314 r1	0:34 1:48 2:10	A:6392785;C:2627202;G:5968712;T:4292408;N:26989	34	48	10	6392785	2627202	5968712	4292408	26989	SRX27572949	SRS23984602	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34788	34788	SRR32233000	SRX27572948	SRS23984601	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 03C	GSM8775313	source name:6 dpf head\|tissue:6 dpf head\|well:P1 C03\|rtindex:GATACGGAAC\|genotype:GAL4UAS replicate1\|geo loc name:missing\|collection date:missing	Well P1 03C	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 C03\|rtindex:GATACGGAAC\|genotype:GAL4UAS replicate1	GSM8775313	GSM8775313: Well P1 03C; Danio rerio; OTHER	GSM8775313 r1	GSM8775313	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-03C.R1.fastq.gz P1-03C.R2.fastq.gz P1-03C_S19_I1_001.fastq.gz	fastq fastq fastq	310907376.0	3379428.0	GSM8775313 r1	0:34 1:48 2:10	A:71568831;C:20485574;G:28605447;T:41329422;N:223270	34	48	10	71568831	20485574	28605447	41329422	223270	SRX27572948	SRS23984601	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34789	34789	SRR32233001	SRX27572947	SRS23984600	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 03B	GSM8775312	source name:6 dpf head\|tissue:6 dpf head\|well:P1 B03\|rtindex:CTAAGAGAAG\|genotype:GAL4UAS replicate1\|geo loc name:missing\|collection date:missing	Well P1 03B	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 B03\|rtindex:CTAAGAGAAG\|genotype:GAL4UAS replicate1	GSM8775312	GSM8775312: Well P1 03B; Danio rerio; OTHER	GSM8775312 r1	GSM8775312	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-03B.R1.fastq.gz P1-03B.R2.fastq.gz P1-03B_S18_I1_001.fastq.gz	fastq fastq fastq	45819128.0	498034.0	GSM8775312 r1	0:34 1:48 2:10	A:7950763;C:3191236;G:7350319;T:5380702;N:32612	34	48	10	7950763	3191236	7350319	5380702	32612	SRX27572947	SRS23984600	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34790	34790	SRR32233002	SRX27572946	SRS23984599	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 03A	GSM8775311	source name:6 dpf head\|tissue:6 dpf head\|well:P1 A03\|rtindex:GATCTTACGC\|genotype:GAL4UAS replicate1\|geo loc name:missing\|collection date:missing	Well P1 03A	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 A03\|rtindex:GATCTTACGC\|genotype:GAL4UAS replicate1	GSM8775311	GSM8775311: Well P1 03A; Danio rerio; OTHER	GSM8775311 r1	GSM8775311	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-03A.R1.fastq.gz P1-03A.R2.fastq.gz P1-03A_S17_I1_001.fastq.gz	fastq fastq fastq	637838760.0	6933030.0	GSM8775311 r1	0:34 1:48 2:10	A:143046137;C:43682277;G:61320584;T:84263744;N:472698	34	48	10	143046137	43682277	61320584	84263744	472698	SRX27572946	SRS23984599	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34791	34791	SRR32233003	SRX27572945	SRS23984598	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 01H	GSM8775302	source name:6 dpf head\|tissue:6 dpf head\|well:P1 H01\|rtindex:AAGCCAGTTA\|genotype:GAL4 replicate1\|geo loc name:missing\|collection date:missing	Well P1 01H	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 H01\|rtindex:AAGCCAGTTA\|genotype:GAL4 replicate1	GSM8775302	GSM8775302: Well P1 01H; Danio rerio; OTHER	GSM8775302 r1	GSM8775302	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-01H.R1.fastq.gz P1-01H.R2.fastq.gz P1-01H_S8_I1_001.fastq.gz	fastq fastq fastq	460.0	5.0	GSM8775302 r1	0:34 1:48 2:10	A:117;C:22;G:25;T:76;N:0	34	48	10	117	22	25	76	0	SRX27572945	SRS23984598	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34792	34792	SRR32233004	SRX27572944	SRS23984597	SRP561592	PRJNA1219235	Neurobehavioral phenotypes in the pan neuronal GAL4 zebrafish line Tgelavl3:KalTA4	GSE288775	Transcriptome Analysis	The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish the Tgelavl3:KalTA4 HuC line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tgelavl3:KalTA4 zebrafish larvae compared to wildtype siblings which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tgelavl3:KalTA4 larvae showed alterations in brain activity brain morphology and behavior including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA seq analysis revealed massive dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis we used economical low pass whole genome sequencing to map the Tol2 mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga a known cell cycle regulator is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan neuronal lines include the length of the elavl3 HuC promoter 9 kb and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines we cloned five neuronal promoters atp6v0cb elavl3 rtn1a sncb and stmn1b ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase based pIGLET system. Our study highlights the importance of using appropriate genetic controls and establishes a roadmap for identifying positional effects in new transgenic lines. Overall design: Tgelavl3:KalTA4 zebrafish were crossed with Tg4xUAS hmgn6 zebrafish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf heads were collected for sci RNA seq.	Well P1 01G	GSM8775301	source name:6 dpf head\|tissue:6 dpf head\|well:P1 G01\|rtindex:CATTCAATCA\|genotype:GAL4 replicate1\|geo loc name:missing\|collection date:missing	Well P1 01G	Demultiplexing trimming alignment filtering and gene count processing were performed on the UMass Chan High Performance Computing cluster using scripts available from https://github.com/bethmartin/sci RNA seq3 pipeline/tree/master. Reads were aligned to GRCz11 release 104 using the Zebrafish Transcriptome Annotation version 4.3.2 Lawson et al. 2020. Assembly: GRCz11 Supplementary files format and content: RDS containing Seurat object with cell level features counts and barcodes Library strategy: sci RNA seq	6 dpf head	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	Sci RNA seq was conducted following the “Tiny Sci” protocol Martin et al. 2023. Briefly Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype elavl3:KalTA4 4xUAS hmgn6 and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf larvae were anesthetized with MS 222 and heads were removed and frozen on dry ice. The remaining body was saved for genotyping.	tissue:6 dpf head\|well:P1 G01\|rtindex:CATTCAATCA\|genotype:GAL4 replicate1	GSM8775301	GSM8775301: Well P1 01G; Danio rerio; OTHER	GSM8775301 r1	GSM8775301	1	tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes Eppendorf 002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP Thermo Fisher 22586 nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf 0030129512. Reverse transcription ligation final distribution second strand synthesis protease digestion tagmentation PCR amplification and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription ligation and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1 10 cycles for Index and 48 cycles for Read2.	OTHER	TRANSCRIPTOMIC	other	PAIRED	ILLUMINA	NextSeq 500	SRP561592	loader:fastq load.py	P1-01G.R1.fastq.gz P1-01G.R2.fastq.gz P1-01G_S7_I1_001.fastq.gz	fastq fastq fastq	61910756.0	672943.0	GSM8775301 r1	0:34 1:48 2:10	A:11472187;C:4248935;G:8848756;T:7686532;N:44854	34	48	10	11472187	4248935	8848756	7686532	44854	SRX27572944	SRS23984597	SRA2070568	UMass Chan	UMass Chan	B	usable mapping rate	illumina	nextseq	unknown	other	unknown	sc	single_cell_plate	scirnaseq	United States	2025-02-04	Larval	Larval	Head	Nervous System
34793	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	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	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	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	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	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	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	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	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	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	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	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	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	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

Advanced export

JSON shape: default, array, newline-delimited

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