Mouse TU tagging: a chemical/genetic intersectional method for purifying cell type-specific nascent RNA
- Leslie Gay1,2,3,
- Michael R. Miller1,2,3,
- P. Britten Ventura2,
- Vidusha Devasthali2,
- Zer Vue4,
- Heather L. Thompson4,
- Sally Temple5,
- Hui Zong2,
- Michael D. Cleary4,
- Kryn Stankunas2 and
- Chris Q. Doe1,2,3,6
- 1Institute of Neuroscience,
- 2Institute of Molecular Biology,
- 3Howard Hughes Medical Institute, University of Oregon, Eugene, Oregon 97403, USA;
- 4School of Natural Sciences, University of California at Merced, Merced, California 95340, USA;
- 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.
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Footnotes
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↵6 Corresponding author
E-mail cdoe{at}uoneuro.uoregon.edu
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Supplemental material is available for this article.
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Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.205278.112.
- Received September 5, 2012.
- Accepted November 19, 2012.
- Copyright © 2013 by Cold Spring Harbor Laboratory Press
Freely available online through the Genes & Development Open Access option.