{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Leo Zeef"],"organism":["Homo sapiens"],"software":["Bcl2fastq-1.8.3"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-7377"],"description":["Melanoma is notorious for its phenotype heterogeneity, which poses a great challenge for current therapies. Two major melanoma-phenotypes are represented by ‘non-EMT-like’ MITFhigh and ‘EMT-like’ MITFlow subpopulations, and while it is thought that transcriptional plasticity leading to a switch from one phenotype to the other drives melanoma progression, recent findings suggest that phenotype switching might not be so clear-cut in vivo. To understand the impact of MITFhigh and MITFlow cells on each other within a heterogeneous tumour we sorted the individual populations from homogeneous and heterogeneous tumours and performed RNAseq.  We analysed MITFhigh and MITFlow subpopulation throughout melanoma progression and found no clear ‘switch’, but rather an ‘adaptation’ with an overlap of phenotype-defining signatures during different stages of melanoma development. Overall, when compared to homogeneous tumours, heterogeneous tumours displayed major changes in ECM-interactions with increased expression of FN1, TNC and matrix re-modellers, and alterations in the repertoire of collagens and adhesion receptors"],"repository":["biostudies-arrayexpress"],"sample_protocol":["Nucleic Acid Extraction - RNA was extracted using an Rneasy Plus Micro Kit (Qiagen).","Library Construction - Strand-specific RNA-seq libraries were prepared using the Illumina workflow with the TruSeq® Stranded mRNA Sample Preparation Kit.","Sequencing - Paired-end reads of 65bp are generated from each sample on the platform of HiSEQ4000.","Sample Collection - 5 million melanoma cells were injected subcutaneously on the back of CD1 nude female mice. Tumours were grown to 1000mm3, following which animals were culled by overdoes of anaesthesia following cardiac puncture and tumours were harvested. For isolation of cells from whole tumours, the tumours were cross chopped with a scalpel as small as possible and then dissociated with pre-warmed Liberase (Roche) at 50ug/ml inserum free media in a humidified incubator at 37oC for 120mins. Serum containing mediawas then added, to neutralise the enzymes, and the samples were centrifuged and resuspendedin PBS for FACS sorting.     Blood was removed by cardiac puncture while mice were under anesthesia for isolating circulatory tumour cells (CTCs). The blood was then diluted in PBS and separated by density gradient centrifugation using Ficoll-Paque (GE Healthcare). The plasma and PBMC layer was then carefully removed and diluted in PBS for FACS sorting.    Cell sorting from tumours was performed using a BD Biosciences FACS Aria Fusion with the excitation laser 488nm and the emission filter 530/30nm bandpass for GFP and excitationlaser 561nm and the emission filter 610/20nm bandpass for mCherry. The gating was set asfollows: FCS-H/FSC-A was used to exclude any events that could represent more than 1 celland FSC/SSC were used to gate out debris based on size. GFP/mCHerry was used to selectGFP or mCherry positive cells. Immediately after FACS sorting sampleswere centrifuged, PBS removed and the cells were thoroughly resuspended in Qiazol."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - DESeq2","Sequence Alignment - Star"],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina HiSeq 4000"],"study_type":["RNA-seq of coding RNA"],"species":["Homo sapiens"],"pubmed_authors":["Leo Zeef","Ping Wang"],"additional_accession":[]},"is_claimable":false,"name":"RNA-Seq of 501mel and WM-266-4 melanoma cell lines transplanted into mice","description":"Melanoma is notorious for its phenotype heterogeneity, which poses a great challenge for current therapies. Two major melanoma-phenotypes are represented by ‘non-EMT-like’ MITFhigh and ‘EMT-like’ MITFlow subpopulations, and while it is thought that transcriptional plasticity leading to a switch from one phenotype to the other drives melanoma progression, recent findings suggest that phenotype switching might not be so clear-cut in vivo. To understand the impact of MITFhigh and MITFlow cells on each other within a heterogeneous tumour we sorted the individual populations from homogeneous and heterogeneous tumours and performed RNAseq.  We analysed MITFhigh and MITFlow subpopulation throughout melanoma progression and found no clear ‘switch’, but rather an ‘adaptation’ with an overlap of phenotype-defining signatures during different stages of melanoma development. Overall, when compared to homogeneous tumours, heterogeneous tumours displayed major changes in ECM-interactions with increased expression of FN1, TNC and matrix re-modellers, and alterations in the repertoire of collagens and adhesion receptors","dates":{"release":"2025-11-23T00:00:00Z","modification":"2025-11-23T02:02:08.152Z","creation":"2022-01-28T17:55:46.364Z"},"accession":"E-MTAB-7377","cross_references":{"ENA":["ERP112144"],"EFO":["EFO_0002944","EFO_0004170","EFO_0004917","EFO_0003816","EFO_0005518","EFO_0003738","EFO_0004184"]}}