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ScRNA-Seq of Fresh Mouse Glioblastoma Cells and Mouse Glioblastoma Cell line

ABSTRACT: Methods: In our study, we added tdTomato gene inherent in our mouse model gene list to obtain the final matrics by modifying the reference genome. Finally, we separated the matrix by the cell type judgment result obtained by the cellranger. Cells with high quality were selected with the following criteria: (1) Genes detected in < 3 cells were removed; (2) Cells with unique molecular identified (UMI) ≤ 150 or ≥ 4500 were removed. (3) Cells with ≥ 20% mitochondrial counts were removed. Results: scRNA-seq for the cells dissociated from a CKO_NG2-CreER tumor (Figure 1B) identified 12 distinct clusters, visualized using t-distributed stochastic neighbor embedding (t-SNE). Referring to known cellular markers in the brain 24, we could identify the cell identities/states of 10 clusters, including two for OPCs (C5 and C7), two for microglia (C1 and C4), one each for astrocytes (C2), endothelial cells (C3), oligodendrocytes (C6), pericytes (C9), neurons (C11) and T/dendritic cells (C12). Projection of lineage marker tdTomato onto the t-SNE map further revealed that 4 clusters (C2, C6, C7 and C9) were likely derived from NG2-CreER labeled cells and/or their progeny Conclusions: scRNA-seq suggests that tumor OPCs in adult OPC-originated gliomas may function as TICs given their strong stemness feature.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE158175 | GEO | 2020/09/18

REPOSITORIES: GEO

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Project description:Background: Dystrophic epidermolysis bullosa (DEB) is a skin blistering disease caused by mutations in COL7A1, which encodes type VII collagen (C7). There is no cure for DEB, but previous work has shown potential therapeutic benefit in increasing production of even partially functional C7. Genome-wide screens using CRISPR-Cas9 have enabled the identification of genes involved in cancer development, drug resistance, and other genetic diseases, suggesting that they could be used to identify novel drivers of C7 production. Methods: A keratinocyte C7 reporter cell line was created by integrating a tdTomato fluorescent marker into the last exon of the endogenous COL7A1 gene. A genome-wide CRISPR activation (CRISPRa) screen was performed with the C7_tdTomato reporter to identify genes and pathways that increase C7 expression. High tdTomato-expressing cells were sorted and sequenced to identify the single guide RNAs (sgRNAs) that became enriched relative to the starting material. Pathway analysis was performed on the corresponding genes to identify regulators and pathways that influence C7 expression. A targeted drug screen was performed in three different keratinocyte cell lines based on the CRISPRa screen results, and C7 upregulation was evaluated. Results: The C7_tdTomato cell line was validated as an effective reporter cell line for detection of C7 upregulation. The CRISPRa screen identified two genes, DENND4B and TYROBP as top hits based on enrichment in the high fluorescence population and representation with multiple sgRNAs. Pathway analysis of the CRISPRa screen showed enrichment of toll-like receptor and interferon-related upstream regulators and functions related to calcium uptake and immune signaling. Several compounds in the targeted drug screen increased C7 expression in at least one keratinocyte line, but only kaempferol, a plant flavonoid, significantly increased C7 mRNA and protein in a DEB patient line. Conclusions: The novel C7_tdTomato reporter cell line can be used to screen compounds for increased C7 expression. The CRISPRa screen combined with a fluorescent reporter cell line can be used to reveal mechanistic regulators of gene expression and therapeutic targets for rare genetic diseases. Kaempferol has shown promising results in increasing C7 production and should be further evaluated as a potential therapeutic for DEB patients.

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ScRNA-Seq of Fresh Mouse Glioblastoma Cells and Mouse Glioblastoma Cell line

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