Applying thiouracil tagging to mouse transcriptome analysis

• Published in: Nature protocols Vol. 9; no. 2; pp. 410 - 420
• Main Authors: Gay, Leslie, Karfilis, Kate V, Miller, Michael R, Doe, Chris Q, Stankunas, Kryn
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.02.2014; Nature Publishing Group
• Subjects: 631/1647/2217/2018; 631/1647/48; 631/208/212/2019; Analytical Chemistry; Animals; Bioinformatics; Biological Techniques; Cell lineage; Computational Biology - methods; Computational Biology/Bioinformatics; Gene expression; Gene Expression Profiling - methods; Gene regulation; Gene sequencing; Labeling; Libraries; Life Sciences; Marking; Methods; Mice; Mice, Transgenic; Microarrays; Organic Chemistry; Physiology; Properties; protocol; Purification; Ribonucleic acid; Ribosomes; RNA; RNA - isolation & purification; RNA - metabolism; RNA sequencing; Technology assessment; Thiouracil - metabolism; Transcription; Transcriptomes; Transgenic mice; Trauma; Uracil; Workflow; Oregon; United States > US
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2014.023

Transcriptional profiling is a powerful approach for studying mouse development, physiology and disease models. Here we describe a protocol for mouse thiouracil tagging (TU tagging), a transcriptome analysis technology that includes in vivo covalent labeling, purification and analysis of cell type–specific RNA. TU tagging enables the isolation of RNA from a given cell population of a complex tissue, avoiding transcriptional changes induced by cell isolation trauma, as well as the identification of actively transcribed RNAs and not preexisting transcripts. Therefore, in contrast to other cell-specific transcriptional profiling methods based on the purification of tagged ribosomes or nuclei, TU tagging provides a direct examination of transcriptional regulation. We describe how to (i) deliver 4-thiouracil to transgenic mice to thio-label cell lineage–specific transcripts, (ii) purify TU-tagged RNA and prepare libraries for Illumina sequencing and (iii) follow a straightforward bioinformatics workflow to identify cell type–enriched or differentially expressed genes. Tissue containing TU-tagged RNA can be obtained in 1 d, RNA-seq libraries can be generated within 2 d and, after sequencing, an initial bioinformatics analysis can be completed in 1 additional day.