Genome-Wide Analysis of DNA Methylation in Single Cells Using a Post-bisulfite Adapter Tagging Approach

  • Heather J. LeeEmail author
  • Sébastien A. SmallwoodEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1712)


DNA methylation is an epigenetic mark implicated in the regulation of key biological processes. Using high-throughput sequencing technologies and bisulfite-based approaches, it is possible to obtain comprehensive genome-wide maps of the mammalian DNA methylation landscape with a single-nucleotide resolution and absolute quantification. However, these methods were only applicable to bulk populations of cells. Here, we present a protocol to perform whole-genome bisulfite sequencing on single cells (scBS-Seq) using a post-bisulfite adapter tagging approach. In this method, bisulfite treatment is performed prior to library generation in order to both convert unmethylated cytosines and fragment DNA to an appropriate size. Then DNA fragments are pre-amplified with concomitant integration of the sequencing adapters, and libraries are subsequently amplified and indexed by PCR. Using scBS-Seq we can accurately measure DNA methylation at up to 50% of individual CpG sites and 70% of CpG islands.

Key words

DNA methylation High-throughput sequencing Bisulfite sequencing Single cell Epigenetics 


  1. 1.
    Smith ZD, Meissner A (2013) DNA methylation: roles in mammalian development. Nat Rev Genet 14:204–220CrossRefPubMedGoogle Scholar
  2. 2.
    Ferguson-Smith AC (2011) Genomic imprinting: the emergence of an epigenetic paradigm. Nat Rev Genet 12:565–575CrossRefPubMedGoogle Scholar
  3. 3.
    Smallwood SA, Kelsey G (2012) De novo DNA methylation: a germ cell perspective. Trends Genet 28:33–42CrossRefPubMedGoogle Scholar
  4. 4.
    Seisenberger S, Peat JR, Hore TA et al (2012) Reprogramming DNA methylation in the mammalian life cycle: building and breaking epigenetic barriers. Philos Trans R Soc Lond Ser B Biol Sci 368:20110330CrossRefGoogle Scholar
  5. 5.
    Harris RA, Wang T, Coarfa C et al (2010) Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications. Nat Biotechnol 28:1097–1105CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Bernstein BE, Stamatoyannopoulos JA, Costello JF et al (2010) The NIH roadmap Epigenomics mapping consortium. Nat Biotechnol 28(10):1045–1048CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    ENCODE Project Consortium (2004) The ENCODE (ENCyclopedia Of DNA Elements) project. Science 306(5696):636–640CrossRefGoogle Scholar
  8. 8.
    Hon GC, Rajagopal N, Shen Y et al (2013) Epigenetic memory at embryonic enhancers identified in DNA methylation maps from adult mouse tissues. Nat Genet 45(10):1198–1206CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Xie W, Schultz MD, Lister R et al (2013) Epigenomic analysis of multilineage differentiation of human embryonic stem cells. Cell 153(5):1134–1148CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Smallwood SA, Tomizawa S-I, Krueger F et al (2011) Dynamic CpG island methylation landscape in oocytes and preimplantation embryos. Nat Genet 43:811–814CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Smallwood SA, Kelsey G (2012) Genome-wide analysis of DNA methylation in low cell numbers by reduced representation bisulfite sequencing. In: Genomic imprinting. Humana Press, Totowa, NJ, pp 187–197CrossRefGoogle Scholar
  12. 12.
    Miura F, Enomoto Y, Dairiki R et al (2012) Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging. Nucleic Acids Res 40:e136CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Shirane K, Toh H, Kobayashi H et al (2013) Mouse oocyte methylomes at base resolution reveal genome-wide accumulation of non-CpG methylation and role of DNA methyltransferases. PLoS Genet 9:e1003439CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Kobayashi H, Sakurai T, Imai M et al (2012) Contribution of intragenic DNA methylation in mouse gametic DNA methylomes to establish oocyte-specific heritable marks. PLoS Genet 8:e1002440CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Peat JR, Dean W, Clark SJ et al (2014) Genome-wide bisulfite sequencing in zygotes identifies demethylation targets and maps the contribution of TET3 oxidation. Cell Rep 9(6):1990–2000CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Smallwood SA, Lee HJ, Angermueller C et al (2014) Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nat Methods 11(8):817–820CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Nashun B, Hill PW, Smallwood SA et al (2015) Continuous histone replacement by Hira is essential for normal transcriptional regulation and de novo DNA methylation during mouse oogenesis. Mol Cell 60(4):611–625CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Quail MA, Otto TD, Gu Y et al (2012) Optimal enzymes for amplifying sequencing libraries. Nat Methods 9:10–11CrossRefGoogle Scholar
  19. 19.
    Krueger F, Andrews SR (2011) Bismark: a flexible aligner and methylation caller for bisulfite-Seq applications. Bioinformatics 27:1571–1572CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Epigenetics Programme, Babraham InstituteCambridgeUK
  2. 2.School of Biomedical Sciences and Pharmacy, Faculty of Health and MedicineThe University of NewcastleCallaghanAustralia
  3. 3.Friedrich Miescher Institute for Biomedical ResearchBaselSwitzerland
  4. 4.Epigenetics Programme, Babraham InstituteCambridgeUK

Personalised recommendations