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MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing

Nature Protocols volume 10, pages 475–483 (2015)Cite this article

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Abstract

Current high-throughput DNA sequencing technologies enable acquisition of billions of data points through which myriad biological processes can be interrogated, including genetic variation, chromatin structure, gene expression patterns, small RNAs and protein–DNA interactions. Here we describe the MethylC-sequencing (MethylC-seq) library preparation method, a 2-d protocol that enables the genome-wide identification of cytosine DNA methylation states at single-base resolution. The technique involves fragmentation of genomic DNA followed by adapter ligation, bisulfite conversion and limited amplification using adapter-specific PCR primers in preparation for sequencing. To date, this protocol has been successfully applied to genomic DNA isolated from primary cell culture, sorted cells and fresh tissue from over a thousand plant and animal samples.

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Figure 1: MethylC-seq library preparation protocol overview.

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Acknowledgements

This work was supported by the Howard Hughes Medical Institute and the Gordon and Betty Moore Foundation (GMBF3034) and grants from the National Science Foundation (MCB1344299 and MCB122246) to J.R.E. R.L. was supported with an Australian Research Council Future Fellowship (FT120100862). R.J.S. was supported by the US National Institutes of Health (R00GM100000) and the US National Science Foundation (IOS-1339194).

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Authors and Affiliations

  1. Genomic Analysis Laboratory, The Salk Institute for Biological Studies, La Jolla, California, USA

    Mark A Urich, Joseph R Nery & Joseph R Ecker

  2. Australian Research Council Center of Excellence in Plant Energy Biology, The University of Western Australia, Perth, Australia

    Ryan Lister

  3. Department of Genetics, University of Georgia, Athens, Georgia, USA

    Robert J Schmitz

  4. Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, California, USA

    Joseph R Ecker

Authors
  1. Mark A Urich
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  2. Joseph R Nery
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Contributions

R.L. and J.R.E. conceived and designed the original protocol. M.A.U., J.R.N., R.L. and R.J.S modified and updated the protocol to its current state. M.A.U., R.L., R.J.S. and J.R.E. wrote the manuscript.

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Correspondence to Joseph R Ecker.

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The authors declare no competing financial interests.

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Urich, M., Nery, J., Lister, R. et al. MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing. Nat Protoc 10, 475–483 (2015). https://doi.org/10.1038/nprot.2014.114

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