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Chromatin immunoprecipitation (ChIP) of plant transcription factors followed by sequencing (ChIP-SEQ) or hybridization to whole genome arrays (ChIP-CHIP)

Nature Protocols volume 5, pages 457–472 (2010)Cite this article

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Abstract

Chromatin immunoprecipitation (ChIP) is a powerful technique to study interactions between transcription factors (TFs) and DNA in vivo. For genome-wide de novo discovery of TF-binding sites, the DNA that is obtained in ChIP experiments needs to be processed for sequence identification. The sequences can be identified by direct sequencing (ChIP-SEQ) or hybridization to microarrays (ChIP-CHIP). Given the small amounts of DNA that are usually obtained in ChIP experiments, successful and reproducible sample processing is challenging. Here we provide a detailed procedure for ChIP of plant TFs, as well as protocols for sample preparation for ChIP-SEQ and for ChIP-CHIP. Our ChIP procedure is optimized for high signal-to-noise ratio starting with tissue fixation, followed by nuclei isolation, immunoprecipitation, DNA amplification and purification. We also provide a guide for primary data analysis of ChIP-SEQ data. The complete protocol for ChIP-SEQ/ChIP-CHIP sample preparation starting from plant harvest takes ∼7 d.

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Figure 1: Chromatin immunoprecipitation (ChIP) and ChIP-SEQ procedures and results.
Figure 2: Estimating the minimum number of hits per nucleotide position based on SOLEXA sequence information from both DNA strands.
Figure 3: Anticipated results of different steps in the ChIP-SEQ procedure.

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Acknowledgements

We thank S. de Folter and F. Turck for their initial advice on the ChIP experiments and W. Busch for advice on sample processing. Some parts of the computations were done at the Poznań Supercomputing and Networking Center.

Author information

Authors and Affiliations

  1. Laboratory of Molecular Biology, Wageningen University, Wageningen, The Netherlands

    Kerstin Kaufmann

  2. Applied Bioinformatics, Plant Research International, Wageningen University & Research Centre, Wageningen, The Netherlands

    Jose M Muiño

  3. Fasteris SA, Plan-les-Ouates, Geneva, Switzerland

    Magne Østerås & Laurent Farinelli

  4. Institute of Plant Genetics, Polish Academy of Sciences, Poznań, Poland

    Pawel Krajewski

  5. Bioscience, Plant Research International, Wageningen, The Netherlands

    Gerco C Angenent

  6. Centre for BioSystems Genomics (CBSG), Wageningen, The Netherlands

    Gerco C Angenent

Authors
  1. Kerstin Kaufmann
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  2. Jose M Muiño
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  3. Magne Østerås
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  4. Laurent Farinelli
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  5. Pawel Krajewski
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  6. Gerco C Angenent
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Contributions

K.K. designed and carried out experiments and wrote the paper; J.M.M. analyzed data, developed computational tools and edited the manuscript; M.O. designed and carried out experiments; L.F. supervised experiments; P.K. supervised the data analysis and development of computational tools; and G.C.A. supervised the experiments and analysis and edited the manuscript.

Corresponding author

Correspondence to Kerstin Kaufmann.

Supplementary information

Supplementary Figure 1 | ChIP-seq and ChIP-chip coverage comparison.

Solexa reads were randomly generated from the Arabidopsis genome using MAQ1 and uniquely mapped using SOAP2. Affymetrix 1.0R tiling probes were uniquely mapped using SOAP with the same parameters. The number of Affymetrix probes and Solexa reads representing non-overlapping genomic windows of size 30432 bp was calculated. Different number of solexa reads were simulated, the same number as unique Affymetrix probes (black), 3 times more (green), 3 times less (red). For illustrative reasons only chromosome 1 is shown. ChIP-seq has similar coverage, except near centromeric regions where the coverage is higher. (PDF 90 kb)

(1) Li, H., Ruan, J. & Durbin, R. Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res 18, 1851-8 (2008).

(2) Li, R., Li, Y., Kristiansen, K. & Wang, J. SOAP: short oligonucleotide alignment program. Bioinformatics 24, 713-4 (2008).

Supplementary Figure 2 | Flowchart of different steps in the ChIP-SEQ data analyses, with examples for different general available software packages.

*For some algorithms, the reads are not extended. (PDF 528 kb)

The references associated with this figure are:

(1) Li, H., Ruan, J. & Durbin, R. Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res 18, 1851-8 (2008).

(2) Li, R., Li, Y., Kristiansen, K. & Wang, J. SOAP: short oligonucleotide alignment program. Bioinformatics 24, 713-4 (2008).

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Kaufmann, K., Muiño, J., Østerås, M. et al. Chromatin immunoprecipitation (ChIP) of plant transcription factors followed by sequencing (ChIP-SEQ) or hybridization to whole genome arrays (ChIP-CHIP). Nat Protoc 5, 457–472 (2010). https://doi.org/10.1038/nprot.2009.244

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