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:

A rapid micro chromatin immunoprecipitation assay (ChIP)

Nature Protocols volume 3, pages 1032–1045 (2008)Cite this article

Abstract

Interactions of proteins with DNA mediate many critical nuclear functions. Chromatin immunoprecipitation (ChIP) is a robust technique for studying protein–DNA interactions. Current ChIP assays, however, either require large cell numbers, which prevent their application to rare cell samples or small-tissue biopsies, or involve lengthy procedures. We describe here a 1-day micro ChIP (μChIP) protocol suitable for up to eight parallel histone and/or transcription factor immunoprecipitations from a single batch of 1,000 cells. μChIP technique is also suitable for monitoring the association of one protein with multiple genomic sites in 100 cells. Alterations in cross-linking and chromatin preparation steps also make μChIP applicable to ∼1-mm3 fresh- or frozen-tissue biopsies. From cell fixation to PCR-ready DNA, the procedure takes ∼8 h for 16 ChIPs.

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: Outline of the micro chromatin immunoprecipitation (μChIP) assay.
Figure 2: Chromatin immunoprecipitation (ChIP) from small cell samples and biopsies.
Figure 3: A PCR-based chromatin fragmentation assay.
Figure 4: Micro chromatin immunoprecipitation (μChIP) analysis of the association of histone H3 modifications and of RNA polymerase II (RNAPII) to gene promoters in human embryonal carcinoma NCCIT cells.
Figure 5: Micro chromatin immunoprecipitation (μChIP) analysis of the association of modified histone H3 and RNA polymerase II (RNAPII) to the GAPDH, OCT4 and SLC10A6 promoters in human osteosarcoma biopsies.

Similar content being viewed by others

References

  1. O'Neill, L.P. & Turner, B.M. Histone H4 acetylation distinguishes coding regions of the human genome from heterochromatin in a differentiation-dependent but transcription-independent manner. EMBO J. 14, 3946–3957 (1995).

    Article  CAS  PubMed Central  Google Scholar 

  2. O'Neill, L.P. & Turner, B.M. Immunoprecipitation of chromatin. Methods Enzymol. 274, 189–197 (1996).

    Article  CAS  PubMed Central  Google Scholar 

  3. Collas, P. & Dahl, J.A. Chop it, ChIP it, check it: the current status of chromatin immunoprecipitation. Front. Biosci. 13, 929–943 (2008).

    Article  CAS  PubMed Central  Google Scholar 

  4. Bernstein, B.E. et al. Genomic maps and comparative analysis of histone modifications in human and mouse. Cell 120, 169–181 (2005).

    Article  CAS  PubMed Central  Google Scholar 

  5. Boyer, L.A. et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947–956 (2005).

    Article  CAS  PubMed Central  Google Scholar 

  6. Bernstein, B.E. et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125, 315–326 (2006).

    Article  CAS  Google Scholar 

  7. O'Neill, L.P., VerMilyea, M.D. & Turner, B.M. Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations. Nat. Genet. 38, 835–841 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  8. Nelson, J.D., Denisenko, O., Sova, P. & Bomsztyk, K. Fast chromatin immunoprecipitation assay. Nucleic Acids Res. 34, e2 (2006).

    Article  PubMed Central  Google Scholar 

  9. Loh, Y.H. et al. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat. Genet. 38, 431–440 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  10. Lee, T.I. et al. Control of developmental regulators by Polycomb in human embryonic stem cells. Cell 125, 301–313 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  11. Dahl, J.A. & Collas, P. Q2ChIP, a quick and quantitative chromatin immunoprecipitation assay unravels epigenetic dynamics of developmentally regulated genes in human carcinoma cells. Stem Cells 25, 1037–1046 (2007).

    Article  CAS  PubMed Central  Google Scholar 

  12. Guenther, M.G., Levine, S.S., Boyer, L.A., Jaenisch, R. & Young, R.A. A chromatin landmark and transcription initiation at most promoters in human cells. Cell 130, 77–88 (2007).

    Article  CAS  PubMed Central  Google Scholar 

  13. Acevedo, L.G. et al. Genome-scale ChIP-chip analysis using 10,000 human cells. Biotechniques 43, 791–797 (2007).

    Article  CAS  PubMed Central  Google Scholar 

  14. Attema, J.L. et al. Epigenetic characterization of hematopoietic stem cell differentiation using miniChIP and bisulfite sequencing analysis. Proc. Natl. Acad. Sci USA 104, 12371–12376 (2007).

    Article  CAS  PubMed Central  Google Scholar 

  15. Zhao, X.D. et al. Whole-genome mapping of histone H3 Lys4 and 27 trimethylations reveals distinct genomic compartments in human embryonic stem cells. Cell Stem Cell 1, 286–298 (2007).

    Article  CAS  Google Scholar 

  16. O'Neill, L.P. & Turner, B.M. Immunoprecipitation of native chromatin: NChIP. Methods 31, 76–82 (2003).

    Article  CAS  PubMed Central  Google Scholar 

  17. Hudson, M.E. & Snyder, M. High-throughput methods of regulatory element discovery. Biotechniques 41, 673 675, 677 passim (2006).

    Article  CAS  PubMed Central  Google Scholar 

  18. Dunn, J.J., McCorkle, S.R., Everett, L. & Anderson, C.W. Paired-end genomic signature tags: a method for the functional analysis of genomes and epigenomes. Genet. Eng. (NY) 28, 159–173 (2007).

    Article  CAS  Google Scholar 

  19. Aiba, K., Carter, M.G., Matoba, R. & Ko, M.S. Genomic approaches to early embryogenesis and stem cell biology. Semin. Reprod. Med. 24, 330–339 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  20. Clark, D.J. & Shen, C.H. Mapping histone modifications by nucleosome immunoprecipitation. Methods Enzymol. 410, 416–430 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  21. Négre, N., Lavrov, S., Hennetin, J., Bellis, M. & Cavalli, G. Mapping the distribution of chromatin proteins by ChIP on chip. Methods Enzymol. 410, 316–341 (2006).

    Article  PubMed Central  Google Scholar 

  22. Wu, J., Smith, L.T., Plass, C. & Huang, T.H. ChIP-chip comes of age for genome-wide functional analysis. Cancer Res. 66, 6899–6902 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  23. Bulyk, M.L. DNA microarray technologies for measuring protein-DNA interactions. Curr. Opin. Biotechnol. 17, 422–430 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  24. O'Geen, H., Nicolet, C.M., Blahnik, K., Green, R. & Farnham, P.J. Comparison of sample preparation methods for ChIP-chip assays. Biotechniques 41, 577–580 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  25. Dahl, J.A. & Collas, P. MicroChIP—a rapid micro chromatin immunoprecipitation assay for small cell samples and biopsies. Nucleic Acids Res. 36, e15 (2008).

    Article  PubMed Central  Google Scholar 

  26. Nelson, J.D., Denisenko, O. & Bomsztyk, K. Protocol for the fast chromatin immunoprecipitation (ChIP) method. Nat. Protoc. 1, 179–185 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  27. Flanagin, S., Nelson, J.D., Castner, D.G., Denisenko, O. & Bomsztyk, K. Microplate-based chromatin immunoprecipitation method, Matrix ChIP: a platform to study signaling of complex genomic events. Nucleic Acids Res. 36, e17 (2008).

    Article  PubMed Central  Google Scholar 

  28. Mikkelsen, T.S. et al. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448, 553–560 (2007).

    Article  CAS  PubMed Central  Google Scholar 

  29. Kiermer, V. Embryos and biopsies on the ChIP-ing forecast. Nat. Methods 3, 583 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  30. Lee, T.I., Johnstone, S.E. & Young, R.A. Chromatin immunoprecipitation and microarray-based analysis of protein location. Nat. Protoc. 1, 729–748 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  31. Spencer, V.A., Sun, J.M., Li, L. & Davie, J.R. Chromatin immunoprecipitation: a tool for studying histone acetylation and transcription factor binding. Methods 31, 67–75 (2003).

    Article  CAS  Google Scholar 

  32. Barski, A. et al. High-resolution profiling of histone methylations in the human genome. Cell 129, 823–837 (2007).

    Article  CAS  Google Scholar 

  33. Orlando, V. Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation. Trends Biochem. Sci. 25, 99–104 (2000).

    Article  CAS  PubMed Central  Google Scholar 

  34. Zeng, P.Y., Vakoc, C.R., Chen, Z.C., Blobel, G.A. & Berger, S.L. In vivo dual cross-linking for identification of indirect DNA-associated proteins by chromatin immunoprecipitation. Biotechniques 41, 694 696, 698 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  35. Roh, T.Y., Cuddapah, S., Cui, K. & Zhao, K. The genomic landscape of histone modifications in human T cells. Proc. Natl. Acad. Sci USA 103, 15782–15787 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  36. Håkelien, A.M., Landsverk, H.B., Robl, J.M., Skålhegg, B.S. & Collas, P. Reprogramming fibroblasts to express T-cell functions using cell extracts. Nat. Biotechnol. 20, 460–466 (2002).

    Article  PubMed Central  Google Scholar 

  37. Landsverk, H.B. et al. Reprogrammed gene expression in a somatic cell-free extract. EMBO Rep. 3, 384–389 (2002).

    Article  CAS  PubMed Central  Google Scholar 

  38. Vandesompele, J., De, P.A. & Speleman, F. Elimination of primer-dimer artifacts and genomic coamplification using a two-step SYBR green I real-time RT-PCR. Anal. Biochem. 303, 95–98 (2002).

    Article  CAS  PubMed Central  Google Scholar 

  39. Ito, K. et al. Decreased histone deacetylase activity in chronic obstructive pulmonary disease. N. Engl. J. Med. 352, 1967–1976 (2005).

    Article  CAS  PubMed Central  Google Scholar 

  40. Pollicino, T. et al. Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones. Gastroenterology 130, 823–837 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  41. Zuccato, C. et al. Widespread disruption of repressor element-1 silencing transcription factor/neuron-restrictive silencer factor occupancy at its target genes in Huntington's disease. J. Neurosci. 27, 6972–6983 (2007).

    Article  CAS  Google Scholar 

  42. Ge, W. et al. Coupling of cell migration with neurogenesis by proneural bHLH factors. Proc. Natl. Acad. Sci USA 103, 1319–1324 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  43. Huang, Y., Doherty, J.J. & Dingledine, R. Altered histone acetylation at glutamate receptor 2 and brain-derived neurotrophic factor genes is an early event triggered by status epilepticus. J. Neurosci. 22, 8422–8428 (2002).

    Article  CAS  Google Scholar 

  44. Le, T.N. et al. Dlx homeobox genes promote cortical interneuron migration from the basal forebrain by direct repression of the semaphorin receptor neuropilin-2. J. Biol. Chem. 282, 19071–19081 (2007).

    Article  CAS  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work is supported by the FUGE, YFF, STAMCELLE and STORFORSK programs of the Research Council of Norway and by the Norwegian Cancer Society.

Author information

Authors and Affiliations

  1. Department of Biochemistry, Faculty of Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, 0317, Norway

    John Arne Dahl & Philippe Collas

Authors
  1. John Arne Dahl
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Philippe Collas
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Philippe Collas.

Ethics declarations

Competing interests

Philippe Collas is a consultant for Diagenode SA, Liege, Belgium

Supplementary information

Supplementary Figure 1

Real-time PCR profiles after analysis of DNA from a 100,000-cell ChIP and of a 100-cell ChIP (PDF 385 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dahl, J., Collas, P. A rapid micro chromatin immunoprecipitation assay (ChIP). Nat Protoc 3, 1032–1045 (2008). https://doi.org/10.1038/nprot.2008.68

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2008.68

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