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

  1. Chris Q. Doe1,2,3,6
  1. 1Institute of Neuroscience,
  2. 2Institute of Molecular Biology,
  3. 3Howard Hughes Medical Institute, University of Oregon, Eugene, Oregon 97403, USA;
  4. 4School of Natural Sciences, University of California at Merced, Merced, California 95340, USA;
  5. 5Neural Stem Cell Institute, Rensselaer, New York 12144, USA

    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.

    Keywords

    Footnotes

    • Received September 5, 2012.
    • Accepted November 19, 2012.

    Freely available online through the Genes & Development Open Access option.

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