{"database": "metadata", "table": "run_metadata", "rows": [[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.", null, null, null, "Well P1 11E", "GSM8775378", null, "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", null, "tiny sci sci RNA seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 \u00b5l lysis buffer B in DNA LowBind tubes Eppendorf  002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP  Thermo Fisher  22586  nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4\u00b0C using a Bioruptor\u00ae Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs  and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf  0030129512. Reverse transcription  ligation  final distribution  second strand synthesis  protease digestion  tagmentation  PCR amplification  and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription  ligation  and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1  10 cycles for Index  and 48 cycles for Read2.", "Sci RNA seq was conducted following the \u201cTiny Sci\u201d protocol Martin et al. 2023. Briefly  Tgelavl3:KalTA4 fish were crossed with Tg4xUAS hmgn6 fish to produce wildtype  elavl3:KalTA4  4xUAS hmgn6  and elavl3:KalTA4; 4xUAS hmgn6 offspring. At 6 dpf  larvae were anesthetized with 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 \u00b5l lysis buffer B in DNA LowBind tubes Eppendorf  002431021. Heads were homogenized with a tissue homogenizer at 1 second intervals for 30 45 seconds until no clumps remained. Following fixation with ice cold methanol and dithiobis succinimidyl propionate DSP  Thermo Fisher  22586  nuclei were resuspended in sucrose PBS TritonX MgCl2 SPBSTM and sonicated for 12 seconds on low at 4\u00b0C using a Bioruptor\u00ae Pico Diagenode. Nuclei were resuspended in SPBSTM with dNTPs  and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate Eppendorf  0030129512. Reverse transcription  ligation  final distribution  second strand synthesis  protease digestion  tagmentation  PCR amplification  and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes Martin et al. 2023. Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription  ligation  and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1  10 cycles for Index  and 48 cycles for Read2.", null, "OTHER", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP561592", null, "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, null, 4748511, 2122090, 5400332, 3588118, 20309, "SRX27572965", "SRS23984618", "SRA2070568", "UMass Chan", "UMass Chan", null, null, null, null, null, null, null, null, null, null, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "other", "unknown", "sc", "single_cell_plate", "scirnaseq", null, "United States", "2025-02-04", "Larval", "Larval", "Head", "Nervous System"]], "columns": ["rowid", "run.accession", "experiment.accession", "sample.accession", "study.accession", "bioproject", "study.title", "study.alias", "study.type", "study.abstract", "study.attributes", "study.PMIDs", "sample.description", "sample.title", "sample.alias", "sample.centername", "sample.attributes", "GEOsample.title", "GEOsample.dataprocessing", "GEOsample.source", "GEOsample.treatmentprotocol", "GEOsample.extractprotocol", "GEOsample.growthprotocol", "GEOsample.characteristics", "GEOsample.accession", "experiment.title", "experiment.alias", "experiment.library_name", "experiment.design_description", "experiment.library_construction_protocol", "experiment.attributes", "experiment.library_strategy", "experiment.library_source", "experiment.library_selection", "experiment.library_layout", "experiment.platform", "experiment.instrument_model", "experiment.spot_descriptor", "experiment.study_ref", "run.title", "run.attributes", "run.filename", "run.semantic_name", "run.total_bases", "run.total_spots", "run.alias", "run.read_lengths", "run.base_counts", "run.r1_length", "run.r2_length", "run.r3_length", "run.r4_length", "run.Acount", "run.Ccount", "run.Gcount", "run.Tcount", "run.Ncount", "run.experiment", "run.pool_member", "submission.accession", "submission.srasource", "submission.bioprojectsource", "seqdetective.n_mates", "seqdetective.mapping_rate.mate1", "seqdetective.mapping_rate.mate2", "seqdetective.nofeature_rate.mate1", "seqdetective.nofeature_rate.mate2", "seqdetective.sparsity.mate1", "seqdetective.sparsity.mate2", "seqdetective.pos_strand_rate.mate1", "seqdetective.pos_strand_rate.mate2", "seqdetective.readlen.mate1", "seqdetective.readlen.mate2", "seqdetective.judgement.mate1", "seqdetective.judgement.mate2", "seqdetective.judgement.reason", "platform_family", "instrument_generation", "read_bias", "selection_class", "prep_kit", "sc_or_bulk", "tech_class", "technology", "tech_variant", "submission.bioprojectsource.country", "earliest_date", "devstage_curation", "devstage_curation_coarse", "tissue_curation", "tissue_curation_coarse"], "primary_keys": ["rowid"], "primary_key_values": ["34771"], "units": {}, "query_ms": 9.546310000587255}