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Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling

Nature Protocols volume 6, pages 468–481 (2011)Cite this article

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Abstract

Genome-wide mapping of 5-methylcytosine is of broad interest to many fields of biology and medicine. A variety of methods have been developed, and several have recently been advanced to genome-wide scale using arrays and next-generation sequencing approaches. We have previously reported reduced representation bisulfite sequencing (RRBS), a bisulfite-based protocol that enriches CG-rich parts of the genome, thereby reducing the amount of sequencing required while capturing the majority of promoters and other relevant genomic regions. The approach provides single-nucleotide resolution, is highly sensitive and provides quantitative DNA methylation measurements. This protocol should enable any standard molecular biology laboratory to generate RRBS libraries of high quality. Briefly, purified genomic DNA is digested by the methylation-insensitive restriction enzyme MspI to generate short fragments that contain CpG dinucleotides at the ends. After end-repair, A-tailing and ligation to methylated Illumina adapters, the CpG-rich DNA fragments (40–220 bp) are size selected, subjected to bisulfite conversion, PCR amplified and end sequenced on an Illumina Genome Analyzer. Note that alignment and analysis of RRBS sequencing reads are not covered in this protocol. The extremely low input requirements (10–300 ng), the applicability of the protocol to formalin-fixed and paraffin-embedded samples, and the technique's single-nucleotide resolution extends RRBS to a wide range of biological and clinical samples and research applications. The entire process of RRBS library construction takes ∼9 d.

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Figure 1: Flowchart of RRBS library construction.
Figure 2: Genomic coverage of typical RRBS libraries.
Figure 3: RRBS library construction.
Figure 4: Adapter sequence and properties.
Figure 5: Guide for preparative size selection on a hypothetical agarose gel.

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Acknowledgements

We thank all the members of Meissner and Gnirke labs, C. Epstein and all the members of the Sequencing Platform at the Broad Institute. C.B. is supported by a Feodor Lynen Fellowship from the Alexander von Humboldt Foundation. A.M. is supported by the Massachusetts Life Science Center, the Pew Charitable Trusts and the NIH Roadmap Initiative on Epigenomics (U01ES017155).

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Authors and Affiliations

  1. Broad Institute, Cambridge, Massachusetts, USA

    Hongcang Gu, Zachary D Smith, Christoph Bock, Patrick Boyle, Andreas Gnirke & Alexander Meissner

  2. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA

    Zachary D Smith, Christoph Bock & Alexander Meissner

  3. Harvard Stem Cell Institute, Cambridge, Massachusetts, USA

    Zachary D Smith, Christoph Bock & Alexander Meissner

  4. Max Planck Institute for Informatics, Saarbrücken, Germany.

    Christoph Bock

Authors
  1. Hongcang Gu
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  2. Zachary D Smith
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  3. Christoph Bock
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  4. Patrick Boyle
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  5. Andreas Gnirke
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  6. Alexander Meissner
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Contributions

H.G., Z.D.S., C.B., P.B., A.G. and A.M. have contributed to the development of the RRBS procedure in the current form. H.G. and A.M. wrote the manuscript with assistance from C.B. and A.G.

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Correspondence to Alexander Meissner.

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The authors declare no competing financial interests.

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Gu, H., Smith, Z., Bock, C. et al. Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling. Nat Protoc 6, 468–481 (2011). https://doi.org/10.1038/nprot.2010.190

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