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Multiplexed locus-specific analysis of DNA methylation in single cells

Nature Protocols volume 10, pages 619–631 (2015)Cite this article

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Abstract

This protocol details a method for measuring the DNA methylation state of multiple target sites in single cells, otherwise known as single-cell restriction analysis of methylation (SCRAM). The basic steps include isolating and lysing single cells, digesting genomic DNA with a methylation-sensitive restriction endonuclease (MSRE) and amplification of multiple targets by two rounds of PCR to determine the methylation status of target sites. The method can reliably and accurately detect the methylation status of multiple target sites in each single cell, and it can be completed in a relatively short time (<2 d) at low cost. Consequently, the method may be preferable over whole-genome methods in applications requiring highly reliable and cost-effective coverage of specific target sites in all cells from a sample and in cases when the DNA methylation states of single CpG sites are representative of the methylation status of corresponding regions of interest.

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Figure 1: Assay principle and workflow for the SCRAM assay.
Figure 2: Workflow for performing qPCR analysis on the Fluidigm 48.48 Dynamic Array and visualization of results.

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Acknowledgements

This work was supported in part by a Visiting Investigator Programme grant (project nos. 092 110 0080 and 1235e00049) from the Joint Council Office of the Singapore Agency for Science, Technology and Research, a Career Development Award (to L.F.C., project no. 14302FG095) from the Joint Council Office of the Singapore Agency for Science, Technology and Research and by the Singapore Institute of Molecular and Cell Biology.

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Authors and Affiliations

  1. Microfluidics Systems Biology Lab, Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore

    Lih Feng Cheow, Stephen R Quake & William F Burkholder

  2. Department of Bioengineering and Department of Applied Physics, Stanford University and Howard Hughes Medical Institute, Stanford, California, USA

    Stephen R Quake

  3. Developmental Epigenetics and Disease Lab, Institute of Molecular and Cell Biology (IMCB), A*STAR, Singapore

    Daniel M Messerschmidt

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  1. Lih Feng Cheow
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Contributions

L.F.C., S.R.Q., W.F.B. and D.M.M. have contributed to the development of the SCRAM assay. L.F.C. wrote the manuscript with assistance from W.F.B. and D.M.M.

Corresponding authors

Correspondence to William F Burkholder or Daniel M Messerschmidt.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Primer organization layout.

The high-throughput nature of the assay requires systematic primer organization. We pre-aliquot 50 µM primer mixes as depicted: For each locus/target (1, 2,..., 24) the two primer combinations (forward-short/reverse [green wells] and forward-long/reverse [blue wells]) are arranged pairwise on a 96-well plate. Each row (A-D) is then systematically transferred to the Assay Inlet ports of the Fluidigm array. Photo courtesy of Fluidigm Corporation.

Supplementary Figure 2 Melting curve analysis.

An exemplary melting curve analysis showing specific amplification in most of the single cell samples (blue arrowhead) and nonspecific amplification in a single sample (red arrowhead).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2 (PDF 252 kb)

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Cheow, L., Quake, S., Burkholder, W. et al. Multiplexed locus-specific analysis of DNA methylation in single cells. Nat Protoc 10, 619–631 (2015). https://doi.org/10.1038/nprot.2015.041

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