{"database": "metadata", "table": "run_metadata", "rows": [[75582, "SRR24658740", "SRX20438404", "SRS17745021", "SRP438621", "PRJNA974396", "Single cell gene expression profiling of prenoplastic cells and myeloid cells during tumour initiation wihin a larval zebrafish model of skin oncogenis driven by inducible HRASG12V", "GSE232900", "Transcriptome Analysis", "Tumour initiation begins with a single oncogenic mutation within a single cell  leading to hyperproliferation and clonal expansion. These mutant preneoplastic cells may undergo various fates such as: death  dormancy  benign growth or malignant growth. Whilst further genetic mutations have been linked to progression of preneoplastic cells  even genetically identical preneoplastic cells may undergo different fates. To better understand the phenotypic heterogeneity between genetically identical preneoplastic cells during tumour initiation we have performed single cell RNA sequencing on preneoplastic cells derived from an inducible larval zebrafish model of skin cancer. Furthermore  inflammation has been identified as a potent driving force in oncogenesis. Macrophages have especially been shown to promote cancer cell growth and progression  whilst neutrophils have the potential to perform either pro  or anti tumour functions. To study the first responses of myeloid cells following initial oncogenic transformation we also profiled sorted myeloid cells at the single cell level. Overall design: Preneoplastic cells and myeloid cells were sorted by FACS from transgenic zebrafish larvae  in which the human oncogene HRASG12V is expressed specifically within epidermal keratinocytes following induction with 4OH Tamoxifen. Induction was carried out at 3dpf. To determine how these preneoplastic cells develop over time within their first day of oncogenic transformation we profiled cells at both 8 and 24 hours post induction. As a control  we also sorted keratinocytes and myeloid cells from time matched larvae which do not express HRASG12V. Preneoplastic cells and keratinocytes were identified by mCherry fluorescence  whilst myeloid cells were identified by EGFP fluorescence from the following larvae: Tgkrtt1c19e:KALTA44 ERT2; UAS:mCherry HRASG12V;mpeg1.1:EGFP and Tgkrtt1c19e:KALTA44 ERT2; UAS:mCherry CAAX;mpeg1.1:EGFP.", null, "pubmed:38272902", null, "HRAS  24hpi  biological replicate 1", "GSM7389056", null, "source name:whole larvae|tissue:whole larvae|cell types:keratinocytes and myeloid cells|strain:Tgkrtt1c19e:KALTA44 ERT2; UAS:mCherry HRASG12V; mpeg1.1:EGFP|treatment:4 OH Tamoxifen|geo loc name:missing|collection date:missing", "HRAS  24hpi  biological replicate 1", "First  each sample was processed individually. The CellRanger pipeline was used to carry out demultiplexing  UMI processing  cell barcode processing  genome alignment  gene counting and identification of empty drops. Removal of ambient RNA was carried out using SoupX. Low quality cells were then removed on the basis of low total counts  low number of detected genes and high percentage of mitochondrial transcripts. Low coverage genes were removed if they were not detected in at least 2 cells. Normalisation was carried out using SCTransform. Principle components and Uniform Manifold Projections UMAP were calculated. A priori markers of keratinocytes  macrophage and neutrophils were inspected visually on UMAPs to aid subsequent clustering. Graph based Louvain clustering was performed and the number of k nearest neighbours varied to capture suitable population as indicated by the expression of a priori markers. Doublets were identified and removed using Scrublet and Doublet Decon. Finally  all samples were aggregated and the data was re normalised and re clustered using the same methods as above. Differential expression analysis between each cluster and the rest of the sample was performed to identify cluster enriched genes Welch\u2019s T test  FDR <0.05  FC > 1.5. The identity of each cluster was confirmed by comparing the cluster enriched genes with the literature and publicly available datasets. Assembly: Danio rerio.GRCz11.101 Supplementary files format and content: Comma separated values files and matrix files", "whole larvae", null, "Whole larvae were dissociated by collagenase and rapid pipetting. Dissociation Solution consisted of HBSS no mag  no cal  plus phenol red  HEPES 15mM   D Glucose 25mM  2% Sterile Goat Serum  Collagenase IV 2.5mg/ml. Groups of 50 larvae were submerged in 2ml of Dissociation Solution prewarmed to 28 degrees and transferred to a 12 well plate. Each well was incubated at 28 degrees with shaking for 5 minutes  followed by 1 minute of rapid pipetting through a P1000 filter tip. Incubation and pipetting were repeated a further two times until a single cell suspension was obtained. For each sample  5 wells were pooled into a single falcon tube  then washed and resuspended in 500ul of Dissociation Solution sans collagenase. Samples were filtered and stained with DAPI shortly prior to sorting using a FACS Fusion Becton Dickson. Keratinocytes/preneoplastic cells and myeloid cells were sorted into a single tube for each sample on the basis of mcherry and EGFP expression respectively. Freshly collected cells were washed once in HBSS at 4 degrees. Cells were counted and checked for viability with a cytometer and approx. 7000 cells were loaded to the 10X chromium chip. Library preparation was performed according to the manufacter\u2019s instructions single cell 3\u2019 v2 protocol  10x Genomics. Briefly  cells were resuspended in master mix and loaded onto the 10X chip alongside partitioning oil and beads to form GEMs gel beads in emulsion. A poly A capture method was used to reverse transcribe each transcript within each GEM whilst adding a Unique Molecular Identifier UMI  cell barcode and Illumina R1 primer sequence. The resulting cDNA was captured on Silane DynaBeads and washed prior to amplification by PCR. To prepare the libraries  further rounds of PCR were carried out  in which the Ilumina R2 primer sequence  paired end constructs with P5 and P7 sequences and a sample index were added.", null, "tissue:whole larvae|cell types:keratinocytes and myeloid cells|strain:Tgkrtt1c19e:KALTA44 ERT2; UAS:mCherry HRASG12V;mpeg1.1:EGFP|treatment:4 OH Tamoxifen", "GSM7389056", "GSM7389056: HRAS  24hpi  biological replicate 1; Danio rerio; RNA Seq", "GSM7389056 r1", "GSM7389056", "1", "Whole larvae were dissociated by collagenase and rapid pipetting. Dissociation Solution consisted of HBSS no mag  no cal  plus phenol red  HEPES 15mM   D Glucose 25mM  2% Sterile Goat Serum  Collagenase IV 2.5mg/ml. Groups of 50 larvae were submerged in 2ml of Dissociation Solution prewarmed to 28 degrees and transferred to a 12 well plate. Each well was incubated at 28 degrees with shaking for 5 minutes  followed by 1 minute of rapid pipetting through a P1000 filter tip. Incubation and pipetting were repeated a further two times until a single cell suspension was obtained. For each sample  5 wells were pooled into a single falcon tube  then washed and resuspended in 500ul of Dissociation Solution sans collagenase. Samples were filtered and stained with DAPI shortly prior to sorting using a FACS Fusion Becton Dickson. Keratinocytes/preneoplastic cells and myeloid cells were sorted into a single tube for each sample on the basis of mcherry and EGFP expression respectively. Freshly collected cells were washed once in HBSS at 4 degrees. Cells were counted and checked for viability with a cytometer and approx. 7000 cells were loaded to the 10X chromium chip. Library preparation was performed according to the manufacter's instructions single cell three prime v2 protocol  10x Genomics. Briefly  cells were resuspended in master mix and loaded onto the 10X chip alongside partitioning oil and beads to form GEMs gel beads in emulsion. A poly A capture method was used to reverse transcribe each transcript within each GEM whilst adding a Unique Molecular Identifier UMI  cell barcode and Illumina R1 primer sequence. The resulting cDNA was captured on Silane DynaBeads and washed prior to amplification by PCR. To prepare the libraries  further rounds of PCR were carried out  in which the Ilumina R2 primer sequence  paired end constructs with P5 and P7 sequences and a sample index were added.", null, "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina NovaSeq 6000", null, "SRP438621", null, null, "H24a_S2_L001_R1_001.fastq.gz H24a_S2_L001_R2_001.fastq.gz", "fastq fastq", 18107807907.0, 152166453.0, "GSM7389056 r1", "0:28 1:91", "A:5035796418;C:3961860116;G:4103474182;T:5006217899;N:459292", 28, 91, null, null, 5035796418, 3961860116, 4103474182, 5006217899, 459292, "SRX20438404", "SRS17745021", "SRA1641488", "University of Edinburgh", "University of Edinburgh", 2, 0.00467, 0.94028, 0.00136, 0.1411, 0.9893, 0.78141, 0.40506, 0.5178, 28, 91, "T", "B", "sc-like readlen", "illumina", "novaseq_era", "unknown", "poly_a", "unknown", "sc", "single_cell_droplet", "10x", null, "United Kingdom", "2023-05-19", "Multi-stage", "Multi-stage", "Whole Organism", "All anatomical structures"]], "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": ["75582"], "units": {}, "query_ms": 12.654777001444018}