Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

The INTACT method for cell type–specific gene expression and chromatin profiling in Arabidopsis thaliana

Nature Protocols volume 6, pages 56–68 (2011)Cite this article

Subjects

Abstract

Genomic studies of cell differentiation and function within a whole organism depend on the ability to isolate specific cell types from a tissue, but this is technically difficult. We developed a method called INTACT (isolation of nuclei tagged in specific cell types) that allows affinity-based isolation of nuclei from individual cell types of a tissue, thereby circumventing the problems associated with mechanical purification techniques. In this method nuclei are affinity-labeled through transgenic expression of a biotinylated nuclear envelope protein in the cell type of interest. Total nuclei are isolated from transgenic plants and biotin-labeled nuclei are then purified using streptavidin-coated magnetic beads, without the need for specialized equipment. INTACT gives high yield and purity of nuclei from the desired cell types, which can be used for genome-wide analysis of gene expression and chromatin features. The entire procedure, from nuclei purification through cDNA preparation or chromatin immunoprecipitation (ChIP), can be completed within 2 d. The protocol we present assumes that transgenic lines are already available, and includes procedural details for amplification of cDNA or ChIP DNA prior to microarray or deep sequencing analysis.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Nuclear targeting fusion protein (NTF) and transgenic lines for the INTACT system.
Figure 2: Purification of tagged nuclei with the INTACT system.

Similar content being viewed by others

References

  1. Mito, Y., Henikoff, J.G. & Henikoff, S. Genome-scale profiling of histone H3.3 replacement patterns. Nat. Genet. 37, 1090–1097 (2005).

    Article  CAS  Google Scholar 

  2. Rao, R.R. & Stice, S.L. Gene expression profiling of embryonic stem cells leads to greater understanding of pluripotency and early developmental events. Biol. Reprod. 71, 1772–1778 (2004).

    Article  CAS  Google Scholar 

  3. Irion, S., Nostro, M.C., Kattman, S.J. & Keller, G.M. Directed differentiation of pluripotent stem cells: from developmental biology to therapeutic applications. Cold Spring Harb. Symp. Quant. Biol. 73, 101–110 (2008).

    Article  CAS  Google Scholar 

  4. Bhattacharya, B., Puri, S. & Puri, R.K. A review of gene expression profiling of human embryonic stem cell lines and their differentiated progeny. Curr. Stem Cell Res. Ther. 4, 98–106 (2009).

    Article  CAS  Google Scholar 

  5. Nakazono, M., Qiu, F., Borsuk, L.A. & Schnable, P.S. Laser-capture microdissection, a tool for the global analysis of gene expression in specific plant cell types: identification of genes expressed differentially in epidermal cells or vascular tissues of maize. Plant Cell 15, 583–596 (2003).

    Article  CAS  Google Scholar 

  6. Jiao, Y. et al. A transcriptome atlas of rice cell types uncovers cellular, functional and developmental hierarchies. Nat. Genet. 41, 258–263 (2009).

    Article  CAS  Google Scholar 

  7. Brunskill, E.W. et al. Atlas of gene expression in the developing kidney at microanatomic resolution. Dev. Cell 15, 781–791 (2008).

    Article  CAS  Google Scholar 

  8. Birnbaum, K. et al. A gene expression map of the Arabidopsis root. Science 302, 1956–1960 (2003).

    Article  CAS  Google Scholar 

  9. Birnbaum, K. et al. Cell type-specific expression profiling in plants via cell sorting of protoplasts from fluorescent reporter lines. Nat. Methods 2, 615–619 (2005).

    Article  CAS  Google Scholar 

  10. Gifford, M.L., Dean, A., Gutierrez, R.A., Coruzzi, G.M. & Birnbaum, K.D. Cell-specific nitrogen responses mediate developmental plasticity. Proc. Natl. Acad. Sci. USA 105, 803–808 (2008).

    Article  CAS  Google Scholar 

  11. Zhang, Y. et al. Identification of genes expressed in C. elegans touch receptor neurons. Nature 418, 331–335 (2002).

    Article  CAS  Google Scholar 

  12. Zhang, C., Barthelson, R.A., Lambert, G.M. & Galbraith, D.W. Global characterization of cell-specific gene expression through fluorescence-activated sorting of nuclei. Plant Physiol. 147, 30–40 (2008).

    Article  CAS  Google Scholar 

  13. Miller, M.R., Robinson, K.J., Cleary, M.D. & Doe, C.Q. TU-tagging: cell type-specific RNA isolation from intact complex tissues. Nat. Methods 6, 439–441 (2009).

    Article  CAS  Google Scholar 

  14. Mustroph, A. et al. Profiling translatomes of discrete cell populations resolves altered cellular priorities during hypoxia in Arabidopsis. Proc. Natl. Acad. Sci. USA 106, 18843–18848 (2009).

    Article  CAS  Google Scholar 

  15. Heiman, M. et al. A translational profiling approach for the molecular characterization of CNS cell types. Cell 135, 738–748 (2008).

    Article  CAS  Google Scholar 

  16. Roy, P.J., Stuart, J.M., Lund, J. & Kim, S.K. Chromosomal clustering of muscle-expressed genes in Caenorhabditis elegans. Nature 418, 975–979 (2002).

    Article  CAS  Google Scholar 

  17. Deal, R.B. & Henikoff, S. A simple method for gene expression and chromatin profiling of individual cell types within a tissue. Dev. Cell 18, 1030–1040 (2010).

    Article  CAS  Google Scholar 

  18. Rose, A. & Meier, I. A domain unique to plant RanGAP is responsible for its targeting to the plant nuclear rim. Proc. Natl. Acad. Sci. USA 98, 15377–15382 (2001).

    Article  CAS  Google Scholar 

  19. Beckett, D., Kovaleva, E. & Schatz, P.J. A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci. 8, 921–929 (1999).

    Article  CAS  Google Scholar 

  20. Barthelson, R.A., Lambert, G.M., Vanier, C., Lynch, R.M. & Galbraith, D.W. Comparison of the contributions of the nuclear and cytoplasmic compartments to global gene expression in human cells. BMC Genomics 8, 340 (2007).

    Article  Google Scholar 

  21. Jacob, Y., Mongkolsiriwatana, C., Veley, K.M., Kim, S.Y. & Michaels, S.D. The nuclear pore protein AtTPR is required for RNA homeostasis, flowering time, and auxin signaling. Plant Physiol. 144, 1383–1390 (2007).

    Article  CAS  Google Scholar 

  22. Meier, I., Xu, X.M., Brkljacic, J., Zhao, Q. & Wang, H.-J. Going green plants' alternative way to position the Ran gradient. J. Microsc. 231, 225–233 (2008).

    Article  CAS  Google Scholar 

  23. An, Y.Q. et al. Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues. Plant J. 10, 107–121 (1996).

    Article  CAS  Google Scholar 

  24. Zilberman, D., Coleman-Derr, D., Ballinger, T. & Henikoff, S. Histone H2A.Z and DNA methylation are mutually antagonistic chromatin marks. Nature 456, 125–129 (2008).

    Article  CAS  Google Scholar 

  25. Ruzicka, D.R., Kandasamy, M.K., McKinney, E.C., Burgos-Rivera, B. & Meagher, R.B. The ancient subclasses of Arabidopsis Actin Depolymerizing Factor genes exhibit novel and differential expression. Plant J. 52, 460–472 (2007).

    Article  CAS  Google Scholar 

  26. Masucci, J.D. et al. The homeobox gene GLABRA2 is required for position-dependent cell differentiation in the root epidermis of Arabidopsis thaliana. Development 122, 1253–1260 (1996).

    CAS  PubMed  Google Scholar 

  27. Gendrel, A.V., Lippman, Z., Martienssen, R. & Colot, V. Profiling histone modification patterns in plants using genomic tiling microarrays. Nat. Methods 2, 213–218 (2005).

    Article  CAS  Google Scholar 

  28. Galbraith, D.W. et al. Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220, 1049–1051 (1983).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. Gehring, F. Steiner and P. Talbert for helpful suggestions on improving the article. This work was supported by funding from the Howard Hughes Medical Institute to S.H. and a Ruth L. Kirschstein Postdoctoral Fellowship from the National Institutes of Health to R.B.D.

Author information

Authors and Affiliations

  1. Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

    Roger B Deal & Steven Henikoff

  2. Howard Hughes Medical Institute, Seattle, Washington, USA

    Steven Henikoff

Authors
  1. Roger B Deal
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Steven Henikoff
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.B.D. developed the protocol with guidance from S.H. The article was written by R.B.D. and edited by S.H.

Corresponding author

Correspondence to Steven Henikoff.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Deal, R., Henikoff, S. The INTACT method for cell type–specific gene expression and chromatin profiling in Arabidopsis thaliana. Nat Protoc 6, 56–68 (2011). https://doi.org/10.1038/nprot.2010.175

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2010.175

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing