{"database": "metadata", "table": "run_metadata", "rows": [[31869, "SRR28735577", "SRX24301539", "SRS21065459", "SRP502616", "PRJNA1101946", "Loss of Stim2 in zebrafish induces retinal gene expression cellular changes resembling glaucoma characteristics [scRNA seq]", "GSE264310", "Transcriptome Analysis", "Calcium is involved in vision processes in retina and is implicated in various pathologies including glaucoma. Rods are protected against prolonged lowering of intracellular calcium ion concentrations by Store Operated Calcium Entry SOCE. We showed zebrafish lacking SOCE calcium sensor Stim2 had problem with vision as indicated by behavior tests  staining of retina and downregulation of genes related to light perception. In this work we aimed to understand the mechanism responsible for the vision problems in stim2 zebrafish knockout. scRNA sequencing of neuronal origin cells from brains of 5 dpf larvae identified 27 clusters. Differently expressed genes were detected in ten clusters including amacrine and GABAergic retinal interneurons and GABAergic optic tectum cells. In five clusters the proportion of cells in stim2 KO fish versus control was significantly decreased including GABAergic diencephalon and optic tectum  and was increased in amacrine and GABAergic retinal interneurons. Transmission Electron Microscopy in stim2 KO fish revealed decrease in width of inner plexiform layer IPL   ganglion cells and their dendrites numbers what is characteristic for glaucoma. Analysis of cell density in the inner nucleus layer  which includes amacrine cells among others  showed a significant decrease in the number of GABAergic neurons.The area of cristae in photoreceptor mitochondria was statistically lower in stim2 KO than in control retinas. Overall design: TgHuC:GCaMP5G zebrafish line and double knockout for stim2 expressing GCaMP5G under the same promoter [stim2a;stim2b /  ; TgHuC:GCaMP5G] were used. Single cell suspensions were obtained by enzymatic and mechanical digestion of 5 dpf dpf larva heads without xxx isolated from two fish lines: stim2 KO 2 replicates   30 larvae per each sample and Tg control  2 replicates  30 larvae per each sample. The cells were sorted based on GCaMP5G fluorescence to obtain the cells of neuronal origin.", "parent bioproject:PRJNA1101942", "pubmed:39424970", null, "Tg  repllicate 1  scRNASeq", "GSM8215879", null, "source name:head|tissue:head|cell type:neuronal|genotype:TgHuC:GCaMP5G|age:5 dpf larvae|geo loc name:missing|collection date:missing", "Tg  repllicate 1  scRNASeq", "Raw reads from fastq files were mapped to the zebrafish reference genome GRCz11 and gene expression were quantified with STARSolo mode of STAR  for each sample with the following parameters:   soloType CB UMI Simple    soloCBwhitelist 3M february 2018.txt [from Cell Ranger]    soloUMIlen 12    soloCBmatchWLtype 1MM multi Nbase pseudocounts    soloUMIfiltering MultiGeneUMI CR    soloUMIdedup 1MM CR    soloCellFilter  EmptyDrops CR Assembly: Danio rerio GRCz11 Supplementary files format and content: Tab separated values files and matrix files", "head", null, "5 dpf larvae were anesthetized by tricaine methanesulfonate MS222  a neuromuscular blocker  and heads were dissected in Dulbecco\u2019s Modified Eagle Medium/Nutrient Mixture F 12 DMEM/F 12; with Ca2+  and L glutamine  and without xxx  and Phenol Red; Gibco with 10% fetal bovine serum FBS; Gibco. Eyes were surgically removed from the fish to focus on the cells of neuronal origins. Single cell suspensions were obtained by enzymatic and mechanical digestion of heads without xxx isolated from two fish lines: stim2 KO and Tg control. Immediately post isolation of single cell suspension  the cells were sorted based on GCaMP5G fluorescence with 488 nm excitation and 510 nm emission to obtain the cells of neuronal origin. The GCaMP5 was expressed under the elav/Huc promoter  which is an early marker of neuronal cells. Cell sorting was performed with a BD FACSAria II BD Biosciences  Franklin Lakes  NJ. Cell viability was measured using the trypan blue staining method  and when the viability of cells was 80%  the cells were immediately loaded on the 10X Chromium system for droplet encapsulation. Approximately 8000 cells were sorted and suspended in 40 \u03bcl volume. Cells were loaded according to the Chromium single cell 3\u2032 kit\u2019s standard protocol V3 chemistry. To prepare the cells for droplet based sequencing  GCaMP5G positive single cell suspension was carefully mixed with reverse transcription mix before loading the cells on the 10X Genomics Chromium system  the standard manufacturer\u2019s protocol. During the encapsulation  the cells were lysed within the droplet  and they released polyadenylated RNA bound to the barcoded bead  which was encapsulated with the cell. Following the 10x Genomics user manual\u2019s guidelines  the droplets were directly subjected to reverse transcription  the emulsion was broken  and cDNA was purified using Silane beads. post the amplification of cDNA with 13 cycles  purification and quantification were performed. The 10X Genomics single cell RNA sequencing library preparation \u2013 involving fragmentation  dA tailing  adapter ligation  and 12 cycle indexing PCR \u2013 was performed.", null, "tissue:head|cell type:neuronal|genotype:TgHuC:GCaMP5G|age:5 dpf larvae", "GSM8215879", "GSM8215879: Tg  repllicate 1  scRNASeq; Danio rerio; RNA Seq", "GSM8215879 r1", "GSM8215879", "1", "5 dpf larvae were anesthetized by tricaine methanesulfonate MS222  a neuromuscular blocker  and heads were dissected in Dulbecco's Modified Eagle Medium/Nutrient Mixture F 12 DMEM/F 12; with Ca2+  and L glutamine  and without xxx  and Phenol Red; Gibco with 10% fetal bovine serum FBS; Gibco. Eyes were surgically removed from the fish to focus on the cells of neuronal origins. Single cell suspensions were obtained by enzymatic and mechanical digestion of heads without xxx isolated from two fish lines: stim2 KO and Tg control. Immediately post isolation of single cell suspension  the cells were sorted based on GCaMP5G fluorescence with 488 nm excitation and 510 nm emission to obtain the cells of neuronal origin. The GCaMP5 was expressed under the elav/Huc promoter  which is an early marker of neuronal cells. Cell sorting was performed with a BD FACSAria II BD Biosciences  Franklin Lakes  NJ. Cell viability was measured using the trypan blue staining method  and when the viability of cells was 80%  the cells were immediately loaded on the 10X Chromium system for droplet encapsulation. Approximately 8000 cells were sorted and suspended in 40 \u03bcl volume. Cells were loaded according to the Chromium single cell 3\u2032 kit's standard protocol V3 chemistry. To prepare the cells for droplet based sequencing  GCaMP5G positive single cell suspension was carefully mixed with reverse transcription mix before loading the cells on the 10X Genomics Chromium system  the standard manufacturer's protocol. During the encapsulation  the cells were lysed within the droplet  and they released polyadenylated RNA bound to the barcoded bead  which was encapsulated with the cell. Following the 10x Genomics user manual's guidelines  the droplets were directly subjected to reverse transcription  the emulsion was broken  and cDNA was purified using Silane beads. post the amplification of cDNA with 13 cycles  purification and quantification were performed. The 10X Genomics single cell RNA sequencing library preparation \u2013 involving fragmentation  dA tailing  adapter ligation  and 12 cycle indexing PCR \u2013 was performed.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 550", null, "SRP502616", null, null, "RIS_F63_0007_P24_V13_S11_R1_001.fastq.gz RIS_F63_0007_P24_V13_S11_R2_001.fastq.gz", "fastq fastq", 143588480.0, 1121785.0, "GSM8215879 r3", "0:28 1:100", "A:41267450;C:30625679;G:32896572;T:38793424;N:5355", 28, 100, null, null, 41267450, 30625679, 32896572, 38793424, 5355, "SRX24301539", "SRS21065459", "SRA1848620", "Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS", "Laboratory of Bioinformatics, Institute of Bioorganic Chemistry PAS", 2, 0.01306, 0.89063, 0.00499, 0.29993, 0.98898, 0.78084, 0.26395, 0.55218, 28, 100, "T", "B", "sc-like readlen", "illumina", "nextseq", "unknown", "cdna_unspecified", "unknown", "sc", "single_cell_droplet", "10x", null, "Poland", "2024-04-18", "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": ["31869"], "units": {}, "query_ms": 7.2139969997806475}