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Nat Protoc. 2015 Apr;10(4):619-31. doi: 10.1038/nprot.2015.041. Epub 2015 Mar 26.

Multiplexed locus-specific analysis of DNA methylation in single cells.

Author information

1
Microfluidics Systems Biology Lab, Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore.
2
1] Microfluidics Systems Biology Lab, Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore. [2] Department of Bioengineering and Department of Applied Physics, Stanford University and Howard Hughes Medical Institute, Stanford, California, USA.
3
Developmental Epigenetics and Disease Lab, Institute of Molecular and Cell Biology (IMCB), A*STAR, Singapore.

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.

PMID:
25811896
DOI:
10.1038/nprot.2015.041
[Indexed for MEDLINE]

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