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Applying thiouracil tagging to mouse transcriptome analysis

Nature Protocols volume 9, pages 410–420 (2014)Cite this article

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Abstract

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.

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Figure 1: Flowchart of the mouse TU-tagging protocol.
Figure 2: DESeq Output plot for visualizing differential expression.

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Acknowledgements

We thank the University of Oregon (UO) Genomics Core Facility for Illumina sequencing; V. Devasthali, U. Hostick and the UO Transgenic Mouse Facility for support generating the CA>GFPstop>HA-UPRT mice; P. Batzel for critical input on the bioinformatics workflow; and B. Simek for beta-testing the bioinformatics protocol. The use of the UO's applied computational instrument for scientific synthesis (ACISS) server was supported by a Major Research Instrumentation grant from the National Science Foundation (no. OCI-0960354). Funding was provided by The US National Institutes of Health (NIH) grant nos. NIH 5R00HL087598 (K.S.) and NIH 3R01DE013085 (Kaartinen, V.S.), and by the Howard Hughes Medical Institute (C.Q.D.).

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Author notes

  1. Michael R Miller

    Present address: Present address: Department of Animal Science, University of California, Davis, Davis, California, USA.,

  2. Leslie Gay and Kate V Karfilis: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA

    Leslie Gay, Kate V Karfilis, Michael R Miller, Chris Q Doe & Kryn Stankunas

  2. Institute of Neuroscience, University of Oregon, Eugene, Oregon, USA

    Leslie Gay, Michael R Miller & Chris Q Doe

  3. Howard Hughes Medical Institute, University of Oregon, Eugene, Oregon, USA

    Leslie Gay & Chris Q Doe

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  1. Leslie Gay
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  2. Kate V Karfilis
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  3. Michael R Miller
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  4. Chris Q Doe
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  5. Kryn Stankunas
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Contributions

L.G., K.V.K., M.R.M., C.Q.D. and K.S. developed the protocol. L.G., K.V.K., C.Q.D. and K.S. prepared and wrote the manuscript.

Corresponding authors

Correspondence to Chris Q Doe or Kryn Stankunas.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Example counts table used in DESeq. The data is for a Type I experiment determining the Tie2:Cre-lineage transcriptome of P6 brains. Data is provided for two pairs of total and TU-tagged RNA samples. (TXT 415 kb)

Supplementary Table 2

Example of DESeq output showing the top 20 most Tie2:Cre-lineage enriched transcripts in P6 whole brain (expression level filtered). The nine enriched positive control endothelial transcripts (of thirteen used) are also shown. (PDF 59 kb)

Supplementary Table 3

Complete DESeq output from the example Type I experiment defining the P6 brain Tie2:Cre lineage transcriptome. (TXT 275 kb)

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Gay, L., Karfilis, K., Miller, M. et al. Applying thiouracil tagging to mouse transcriptome analysis. Nat Protoc 9, 410–420 (2014). https://doi.org/10.1038/nprot.2014.023

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