single-cell dna sequencing mission bio tapestri  (Mission Bio)

Journal: Nature Genetics

Article Title: A combinatorial genetic strategy for exploring complex genotype–phenotype associations in cancer

doi: 10.1038/s41588-024-01674-1

Figure Lengend Snippet: ( a ) Representative flow cytometry plot for the isolation of mouse bladder urothelial (top) and prostate epithelial (bottom) from dissociated tissues based on a Lin - (CD45 - CD31 - Ter119 - ) EpCAM + CD49f high immunophenotype. ( b ) Images of organoid cultures of mouse bladder urothelial and prostate epithelial cells on day 1 and day 5 after seeding. ( c ) Table showing the amplicons represented in a custom Mission Bio Tapestri single-cell DNA amplicon sequencing panel. ( d ) Table showing results of a validation study where a defined mixture of 3T3 cells with an unlabeled population and others labeled with combinations of lentiviruses encoding distinct barcodes were analyzed using the Mission Bio Tapestri single-cell DNA amplicon sequencing panel to determine clonality. ~2,000 cells were analyzed. ( e ) Overview of experiments with infection of mouse prostate epithelial (mPE) cells with a diverse barcoded lentiviral library in organoid culture across a range of multiplicity-of-infection (MOI) and quantification of viral copy number per cell across the population by single-cell amplicon sequencing. Created with BioRender.com .

Article Snippet: Extended Data Fig. 1 Isolation of mouse bladder urothelial and prostate epithelial cells for organoid culture and design/validation of a custom Mission Bio Tapestri single-cell DNA amplicon sequencing panel. ( a ) Representative flow cytometry plot for the isolation of mouse bladder urothelial (top) and prostate epithelial (bottom) from dissociated tissues based on a Lin - (CD45 - CD31 - Ter119 - ) EpCAM + CD49f high immunophenotype. ( b ) Images of organoid cultures of mouse bladder urothelial and prostate epithelial cells on day 1 and day 5 after seeding. ( c ) Table showing the amplicons represented in a custom Mission Bio Tapestri single-cell DNA amplicon sequencing panel. ( d ) Table showing results of a validation study where a defined mixture of 3T3 cells with an unlabeled population and others labeled with combinations of lentiviruses encoding distinct barcodes were analyzed using the Mission Bio Tapestri single-cell DNA amplicon sequencing panel to determine clonality.

Techniques: Flow Cytometry, Isolation, Amplification, Sequencing, Biomarker Discovery, Labeling, Infection

Journal: Nature Genetics

Article Title: A combinatorial genetic strategy for exploring complex genotype–phenotype associations in cancer

doi: 10.1038/s41588-024-01674-1

Figure Lengend Snippet: a , Top, schematic of a lentiviral (LV) construct with matching barcodes (BCs) at the 5′ and 3′ ends. Bottom, overview of experiments with LV infection of primary mouse cells in organoid culture and quantification of transduction. Created with BioRender.com . CMV, cytomegalovirus; UBC, ubiquitin C; WVH8, Woodchuck hepatitis virus 8 post-transcriptional regulatory element. b , Left, brightfield and GFP images of mouse bladder or prostate organoids 72 h after mock or GFP LV transduction. Scale bar, 400 µm. Right, tables summarizing quantification of LV transduction by flow cytometry and LV copies of GFP (± s.d.) by qPCR. c , Left, plot of the distribution of LV copies per mPE cell at different MOIs 72 h after transduction. Right, table summarizing viral copy number (VCN) population frequencies at varying MOIs. The experiment was independently repeated three times with similar results. d , Scheme of the mBU organoid transformation assay to uncover functional genotype–phenotype associations in bladder cancer. Created with BioRender.com . e , Left, gross image of a tumor arising from mBU transformed with a BU-LVP. Middle, low-magnification image of the hematoxylin and eosin (H&E)-stained tumor section. Right, high-magnification images of H&E-stained and immunohistochemically stained sections of regions with distinct histologies. Scale bars, 50 µm. Each FHBT model is a unique tumor that is the result of an independent experiment. f , Clonal architecture of the three dominant clones in the tumor as determined by Mission Bio Tapestri single-cell analysis of LV barcodes. g , Left, tumor tissue section after LCM of the histologically distinct regions. Right, table showing the associations between regional tumor histologies and clones in f based on LCM and bulk DNA amplicon sequencing of LV barcodes.

Article Snippet: Extended Data Fig. 1 Isolation of mouse bladder urothelial and prostate epithelial cells for organoid culture and design/validation of a custom Mission Bio Tapestri single-cell DNA amplicon sequencing panel. ( a ) Representative flow cytometry plot for the isolation of mouse bladder urothelial (top) and prostate epithelial (bottom) from dissociated tissues based on a Lin - (CD45 - CD31 - Ter119 - ) EpCAM + CD49f high immunophenotype. ( b ) Images of organoid cultures of mouse bladder urothelial and prostate epithelial cells on day 1 and day 5 after seeding. ( c ) Table showing the amplicons represented in a custom Mission Bio Tapestri single-cell DNA amplicon sequencing panel. ( d ) Table showing results of a validation study where a defined mixture of 3T3 cells with an unlabeled population and others labeled with combinations of lentiviruses encoding distinct barcodes were analyzed using the Mission Bio Tapestri single-cell DNA amplicon sequencing panel to determine clonality.

Techniques: Construct, Infection, Transduction, Ubiquitin Proteomics, Virus, Flow Cytometry, Transformation Assay, Functional Assay, Staining, Clone Assay, Single-cell Analysis, Amplification, Sequencing

Journal: Nature Genetics

Article Title: A combinatorial genetic strategy for exploring complex genotype–phenotype associations in cancer

doi: 10.1038/s41588-024-01674-1

Figure Lengend Snippet: Schema showing the ( a ) generation of individual lentiviruses from the library in arrayed format with subsequent pooling and concentration by ultracentrifugation and ( b ) transduction of respective mouse bladder urothelial (mBU) or prostate epithelial (mPE) cells in organoid culture with concentrated lentiviral libraries to determine lentiviral barcode representation by bulk amplicon sequencing of genomic DNA. Created with BioRender.com . ( c ) Representative distribution of barcoded lentiviruses within a library with skewed enrichment of shRNA relative to ORF lentiviruses. ( d ) Representative distribution of barcoded lentiviruses within a library after applying information from c to adjust producer cell surface area in a for the generation of the lentiviral library.

Article Snippet: Extended Data Fig. 1 Isolation of mouse bladder urothelial and prostate epithelial cells for organoid culture and design/validation of a custom Mission Bio Tapestri single-cell DNA amplicon sequencing panel. ( a ) Representative flow cytometry plot for the isolation of mouse bladder urothelial (top) and prostate epithelial (bottom) from dissociated tissues based on a Lin - (CD45 - CD31 - Ter119 - ) EpCAM + CD49f high immunophenotype. ( b ) Images of organoid cultures of mouse bladder urothelial and prostate epithelial cells on day 1 and day 5 after seeding. ( c ) Table showing the amplicons represented in a custom Mission Bio Tapestri single-cell DNA amplicon sequencing panel. ( d ) Table showing results of a validation study where a defined mixture of 3T3 cells with an unlabeled population and others labeled with combinations of lentiviruses encoding distinct barcodes were analyzed using the Mission Bio Tapestri single-cell DNA amplicon sequencing panel to determine clonality.

Techniques: Concentration Assay, Transduction, Amplification, Sequencing, shRNA