Applying thiouracil tagging to mouse transcriptome analysis.
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.
1] Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA. [2] Institute of Neuroscience, University of Oregon, Eugene, Oregon, USA. [3] Howard Hughes Medical Institute, University of Oregon, Eugene, Oregon, USA. [4].
Leslie
L
Gay
1] Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA. [2].
Kate V
KV
Karfilis
1] Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA. [2] Institute of Neuroscience, University of Oregon, Eugene, Oregon, USA. [3].
Michael R
MR
Miller
1] Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA. [2] Institute of Neuroscience, University of Oregon, Eugene, Oregon, USA. [3] Howard Hughes Medical Institute, University of Oregon, Eugene, Oregon, USA.
Chris Q
CQ
Doe
Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA.
Kryn
K
Stankunas
D000818:Animals; D019295:Computational Biology / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods*; D051379:Mice; D008822:Mice, Transgenic; D012313:RNA / Q000302:isolation & purification* / Q000378:metabolism; D013889:Thiouracil / Q000378:metabolism
D013889:Thiouracil; D012313:RNA