Protocol | Published:

Methylome analysis using MeDIP-seq with low DNA concentrations

Nature Protocols volume 7, pages 617636 (2012) | Download Citation

Abstract

DNA methylation is an epigenetic mark that has a crucial role in many biological processes. To understand the functional consequences of DNA methylation on phenotypic plasticity, a genome-wide analysis should be embraced. This in turn requires a technique that balances accuracy, genome coverage, resolution and cost, yet is low in DNA input in order to minimize the drain on precious samples. Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) fulfils these criteria, combining MeDIP with massively parallel DNA sequencing. Here we report an improved protocol using 100-fold less genomic DNA than that commonly used. We show comparable results for specificity (>97%) and enrichment (>100-fold) over a wide range of DNA concentrations (5,000–50 ng) and demonstrate the utility of the protocol for the generation of methylomes from rare bone marrow cells using 160–300 ng of starting DNA. The protocol described here, i.e., DNA extraction to generation of MeDIP-seq library, can be completed within 3–5 d.

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Acknowledgements

O.T. was supported by a PhD studentship from the UK Medical Research Council. S.S. was supported by the Boehringer Ingelheim Fonds, the Biotechnology and Biological Sciences Research Council (BBSRC) and Cambridge European Trust. W.R. is a Senior Investigator of the Wellcome Trust, and work in the Reik laboratory was supported by the BBSRC, Medical Research Council (MRC), EU Network of Excellence EpiGenesys, and Cellcentric. Work in the Beck laboratory was supported by the Wellcome Trust (084071), a Royal Society Wolfson Research Merit Award (WM100023), MRC (G1000411), the Engineering and Physical Sciences Research Council (EPSRC) (P14187), Innovative Medicines Initiative—Joint Undertaking (IMI-JU) OncoTrack (115234), and EU Seventh Framework Programme (EU-FP7) projects HEROIC (018883), EPIGENESYS (257082), IDEAL (259679), ITFoM (085602) and BLUEPRINT (282510).

Author information

Affiliations

  1. University College London (UCL) Cancer Institute, University College London, London, UK.

    • Oluwatosin Taiwo
    • , Gareth A Wilson
    • , Tiffany Morris
    • , Stephan Beck
    •  & Lee M Butcher
  2. UCL Institute of Healthy Ageing, University College London, London, UK.

    • Oluwatosin Taiwo
    •  & Daniel Pearce
  3. Laboratory of Developmental Genetics and Imprinting, The Babraham Institute, Cambridge, UK.

    • Stefanie Seisenberger
    •  & Wolf Reik
  4. Centre for Trophoblast Research, University of Cambridge, Cambridge, UK.

    • Wolf Reik

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Contributions

O.T., L.M.B. and S.B. conceived the study. S.S. and W.R. contributed data (early version of the protocol). D.P. contributed materials. O.T. and L.M.B. did the experiments and analyzed data. G.A.W. and T.M. did the bioinformatics analysis. O.T., L.M.B., G.A.W. and S.B. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Oluwatosin Taiwo or Stephan Beck or Lee M Butcher.

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DOI

https://doi.org/10.1038/nprot.2012.012

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