Analyzing Transcription Factor Occupancy During Embryo Development Using ChIP-seq

  • Yad Ghavi-Helm
  • Eileen E. M. Furlong
Part of the Methods in Molecular Biology book series (MIMB, volume 786)


Accurately assessing the binding of transcription factors to cis-regulatory elements in vivo is an essential step toward understanding the mechanisms that govern embryonic development. Genome-wide transcription factor location analysis has been facilitated by the development of high-density tiling arrays (ChIP-on-chip), and more recently by next-generation sequencing technologies, which are used to sequence the DNA fragments obtained from chromatin immunoprecipitation experiments (ChIP-seq). This chapter provides a detailed protocol of the different steps required to generate a successful ChIP-seq library, starting from embryo collection and fixation to chromatin preparation, immunoprecipitation, and finally library preparation. The protocol is optimized for Drosophila embryos, but can be adapted to any organism. The obtained library is suitable for sequencing on an Illumina GAIIx platform.

Key words

Drosophila Transcription Development Chromatin immunoprecipitation ChIP-seq Next-generation sequencing 


  1. 1.
    Bonn, S., and Furlong, E. E. (2008) cis-Regulatory networks during development: a view of Drosophila Curr Opin Genet Dev 18, 513–20.PubMedCrossRefGoogle Scholar
  2. 2.
    Davidson, E. H. (2006) The Regulatory Genome: Gene Regulatory Networks In Development And Evolution Academic Press.Google Scholar
  3. 3.
    Sandmann, T., Jensen, L. J., Jakobsen, J. S., Karzynski, M. M., Eichenlaub, M. P., Bork, P., and Furlong, E. E. (2006) A temporal map of transcription factor activity: mef2 directly regulates target genes at all stages of muscle development Dev Cell 10, 797–807.PubMedCrossRefGoogle Scholar
  4. 4.
    Sandmann, T., Girardot, C., Brehme, M., Tongprasit, W., Stolc, V., and Furlong, E. E. (2007) Genes Dev 21, 436–49.PubMedCrossRefGoogle Scholar
  5. 5.
    Li, X. Y., MacArthur, S., Bourgon, R., Nix, D., Pollard, D. A., Iyer, V. N., Hechmer, A., Simirenko, L., Stapleton, M., Luengo Hendriks, C. L., Chu, H. C., Ogawa, N., Inwood, W., Sementchenko, V., Beaton, A., Weiszmann, R., Celniker, S. E., Knowles, D. W., Gingeras, T., Speed, T. P., Eisen, M. B., and Biggin, M. D. (2008) Transcription factors bind thousands of active and inactive regions in the Drosophila blastoderm PLoS Biol 6, e27.Google Scholar
  6. 6.
    Liu, Y. H., Jakobsen, J. S., Valentin, G., Amarantos, I., Gilmour, D. T., and Furlong, E. E. (2009) A systematic analysis of Tinman function reveals Eya and JAK-STAT signaling as essential regulators of muscle development Dev Cell 16, 280–91.Google Scholar
  7. 7.
    Zeitlinger, J., Zinzen, R. P., Stark, A., Kellis, M., Zhang, H., Young, R. A., and Levine, M. (2007) Whole-genome ChIP-chip analysis of Dorsal, Twist, and Snail suggests integration of diverse patterning processes in the Drosophila embryo Genes Dev 21, 385–90.Google Scholar
  8. 8.
    Jakobsen, J. S., Braun, M., Astorga, J., Gustafson, E. H., Sandmann, T., Karzynski, M., Carlsson, P., and Furlong, E. E. (2007) Temporal ChIP-on-chip reveals Biniou as a universal regulator of the visceral muscle transcriptional network Genes Dev 21, 2448–60.Google Scholar
  9. 9.
    MacArthur, S., Li, X. Y., Li, J., Brown, J. B., Chu, H. C., Zeng, L., Grondona, B. P., Hechmer, A., Simirenko, L., Keranen, S. V., Knowles, D. W., Stapleton, M., Bickel, P., Biggin, M. D., and Eisen, M. B. (2009) Developmental roles of 21 Drosophila transcription factors are determined by quantitative differences in binding to an overlapping set of thousands of genomic regions Genome Biol 10, R80.PubMedCrossRefGoogle Scholar
  10. 10.
    Zinzen, R. P., Girardot, C., Gagneur, J., Braun, M., and Furlong, E. E. (2009) Combinatorial binding predicts spatio-temporal cis-regulatory activity Nature 462, 65–70.Google Scholar
  11. 11.
    Aparicio, O., Geisberg, J. V., and Struhl, K. (2004) Chromatin immunoprecipitation for determining the association of proteins with specific genomic sequences in vivo Curr Protoc Cell Biol 17, Unit 17 7.Google Scholar
  12. 12.
    Sandmann, T., Jakobsen, J. S., and Furlong, E. E. (2006) ChIP-on-chip protocol for genome-wide analysis of transcription factor binding in Drosophila melanogaster embryos Nat Protoc 1, 2839–55.Google Scholar
  13. 13.
    Johnson, D. S., Mortazavi, A., Myers, R. M., and Wold, B. (2007) Genome-wide mapping of in vivo protein-DNA interactions Science 316, 1497–502.Google Scholar
  14. 14.
    Sisson, J. C. (2000) Culturing Large Populations Of Drosophila for Protein Biochemistry Drosophila Protocols.Google Scholar
  15. 15.
    Campos-Ortega, J. A., and Hartenstein, V. (1985) The Embryonic Development of Drosophila melanogaster Springer.Google Scholar
  16. 16.
    Ng, P., Wei, C. L., and Ruan, Y. (2007) Paired-end diTagging for transcriptome and genome analysis Curr Protoc Mol Biol 21, Unit 21 12.Google Scholar
  17. 17.
    Quail, M. A., Kozarewa, I., Smith, F., Scally, A., Stephens, P. J., Durbin, R., Swerdlow, H., and Turner, D. J. (2008) A large genome center’s improvements to the Illumina sequencing system Nat Methods 5, 100510.Google Scholar
  18. 18.
    Park, P. J. (2009) ChIP-seq: advantages and challenges of a maturing technology Nat Rev Genet 10, 669–80.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Genome Biology UnitEuropean Molecular Biology LaboratoryHeidelbergGermany

Personalised recommendations