Multiplexed locus-specific analysis of DNA methylation in single cells

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.