run_metadata: 75585

This data as json

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
75585	SRR24658743	SRX20438401	SRS17745019	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.		pubmed:38272902		CAAX control 24hpi biological replicate 1	GSM7389052		source name:whole larvae\|tissue:whole larvae\|cell types:keratinocytes and myeloid cells\|strain:Tgkrtt1c19e:KALTA44 ERT2; UAS:mCherry CAAX; mpeg1.1:EGFP\|treatment:4 OH Tamoxifen\|geo loc name:missing\|collection date:missing	CAAX control 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’s 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		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 3’ 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.		tissue:whole larvae\|cell types:keratinocytes and myeloid cells\|strain:Tgkrtt1c19e:KALTA44 ERT2; UAS:mCherry CAAX;mpeg1.1:EGFP\|treatment:4 OH Tamoxifen	GSM7389052	GSM7389052: CAAX control 24hpi biological replicate 1; Danio rerio; RNA Seq	GSM7389052 r1	GSM7389052	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.		RNA-Seq	TRANSCRIPTOMIC	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000		SRP438621			C24a_S7_L001_R1_001.fastq.gz C24a_S7_L001_R2_001.fastq.gz	fastq fastq	16830453339.0	141432381.0	GSM7389052 r1	0:28 1:91	A:4705072415;C:3671527815;G:3780778905;T:4672644538;N:429666	28	91			4705072415	3671527815	3780778905	4672644538	429666	SRX20438401	SRS17745019	SRA1641488	University of Edinburgh	University of Edinburgh	2	0.00383	0.93899	0.00124	0.14413	0.99204	0.78388	0.43002	0.50704	28	91	T	B	sc-like readlen	illumina	novaseq_era	unknown	poly_a	unknown	sc	single_cell_droplet	10x		United Kingdom	2023-05-19	Multi-stage	Multi-stage	Whole Organism	All anatomical structures