Mouse TU tagging: a chemical/genetic intersectional method for purifying cell type-specific nascent RNA.

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Public Summary:
We describe the use of a technique, TU-tagging, that allows identification of genes expressed in cells within their normal tissue environment. Stem cells are particularly sensitive to communication with other cells in complex tissues and this technique allows identification of genes expressed within stem cells or their surrounding "niche" cells under normal, physiologic conditions. We used TU-tagging to identify gene expression in endothelial cells of the brain (an important niche population for neural stem cells) and to identify gene expression in immune system cells versus niche cells of the spleen, using a blood stem cell transplantation model.
Scientific Abstract:
Transcriptional profiling is a powerful approach for understanding development and disease. Current cell type-specific RNA purification methods have limitations, including cell dissociation trauma or inability to identify all RNA species. Here, we describe "mouse thiouracil (TU) tagging," a genetic and chemical intersectional method for covalent labeling and purification of cell type-specific RNA in vivo. Cre-induced expression of uracil phosphoribosyltransferase (UPRT) provides spatial specificity; injection of 4-thiouracil (4TU) provides temporal specificity. Only UPRT(+) cells exposed to 4TU produce thio-RNA, which is then purified for RNA sequencing (RNA-seq). This method can purify transcripts from spatially complex and rare (<5%) cells, such as Tie2:Cre(+) brain endothelia/microglia (76% validated by expression pattern), or temporally dynamic transcripts, such as those acutely induced by lipopolysaccharide (LPS) injection. Moreover, generating chimeric mice via UPRT(+) bone marrow transplants identifies immune versus niche spleen RNA. TU tagging provides a novel method for identifying actively transcribed genes in specific cells at specific times within intact mice.