期刊
GENES & DEVELOPMENT
卷 27, 期 1, 页码 98-115出版社
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1101/gad.205278.112
关键词
brain; endothelial cells; heart; microglia; nascent RNA; transcriptional profiling
资金
- NIH/NHLBI [5R00HL087598]
- March of Dimes Basil O'Connor Award
- Department of Defense Peer-Reviewed Cancer Research Program [CA100469]
- Pew Charitable Trust
- NRSA predoctoral fellowship
- California Institute for Regenerative Medicine [RT1-01052-1]
- Empire State Stem Cell Fund from New York State Department of Health [C024352]
- Howard Hughes Medical Institute
- CDMRP [CA100469, 545619] Funding Source: Federal RePORTER
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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