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Genes Dev. 2013 Jan 1;27(1):98-115. doi: 10.1101/gad.205278.112.

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

Author information

1
Institute of Neuroscience, University of Oregon, Eugene, 97403, USA.

Erratum in

  • Genes Dev. 2014 Dec 1;28(23):2677.

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.

PMID:
23307870
PMCID:
PMC3553287
DOI:
10.1101/gad.205278.112
[Indexed for MEDLINE]
Free PMC Article
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