Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-seq)

Abstract

DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome. Here we present a detailed protocol for scBS-seq that includes our most recent developments to optimize recovery of CpGs, mapping efficiency and success rate; reduce hands-on time; and increase sample throughput with the option of using an automated liquid handler. We provide step-by-step instructions for each stage of the method, comprising cell lysis and bisulfite (BS) conversion, preamplification and adaptor tagging, library amplification, sequencing and, lastly, alignment and methylation calling. An individual with relevant molecular biology expertise can complete library preparation within 3 d. Subsequent computational steps require 1–3 d for someone with bioinformatics expertise.

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Figure 1: Overview of scBS-seq library preparation protocol.
Figure 2: Bioanalyzer electropherograms of typical scBS-seq libraries.
Figure 3: Quality control criteria for scBS-seq libraries.

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Acknowledgements

W.R. was supported by the Biotechnology and Biological Sciences Research Council (BBSRC), the Wellcome Trust, the EU Blueprint and EpiGeneSys. G.K. was supported by the BBSRC and the Medical Research Council (MRC). H.J.L. was supported by the EU Network of Excellence (NoE) EpiGeneSys.

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S.J.C. developed the updated protocol and wrote the manuscript with input from all other authors; S.A.S. and H.J.L conceived the original protocol and developed the updated version; F.K. developed the bioinformatics pipeline; and W.R. and G.K. conceived of and jointly supervised the study.

Corresponding authors

Correspondence to Stephen J Clark or Wolf Reik or Gavin Kelsey.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

iPCRTag primer sequences. Sequence obtained is the index that is read by the Illumina machine and is the reverse complement of index portion of the primer. Primers should be ordered HPLC-purified and resuspended in EB to 100 μM. (PDF 299 kb)

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Clark, S., Smallwood, S., Lee, H. et al. Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-seq). Nat Protoc 12, 534–547 (2017). https://doi.org/10.1038/nprot.2016.187

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