title: Mouse TU tagging: a chemical/genetic intersectional method for purifying cell type-specific nascent RNA.
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.
authors: Institute of Neuroscience, University of Oregon, Eugene, 97403, USA.
authors: Leslie
authors: L
authors: Gay
authors: Michael R
authors: MR
authors: Miller
authors: P Britten
authors: PB
authors: Ventura
authors: Vidusha
authors: V
authors: Devasthali
authors: Zer
authors: Z
authors: Vue
authors: Heather L
authors: HL
authors: Thompson
authors: Sally
authors: S
authors: Temple
authors: Hui
authors: H
authors: Zong
authors: Michael D
authors: MD
authors: Cleary
authors: Kryn
authors: K
authors: Stankunas
authors: Chris Q
authors: CQ
authors: Doe
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism; D016026:Bone Marrow Transplantation; D001921:Brain / Q000196:embryology / Q000378:metabolism; D002678:Chimera; D020869:Gene Expression Profiling; D051379:Mice; D008967:Molecular Biology / Q000379:methods*; D012313:RNA / Q000302:isolation & purification*; D013194:Staining and Labeling / Q000379:methods*; D013889:Thiouracil / Q000378:metabolism*; D019076:Transgenes / Q000235:genetics
chemical_list: D013889:Thiouracil; D012313:RNA