单细胞特定位点甲基化修饰研究指南.docx

单细胞特定位点甲基化修饰研究指南2015.3AbstractAbstract? Introduction? Materials? Procedure? Troubleshooting? Timing? Anticipated results? References? Acknowledgments? Author information? Supplementary informationThis 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.IntroductionMethylation of ?CpGdinucleotides is a highly prevalent DNA modification in mammalian genomes that has important roles in epigenetic regulation of gene expression1, genomic imprinting2, repression of transposable elements3 and X-chromosome inactivation1. The precise timing and dynamics of these processes are important for proper development4. Nevertheless, it is challenging to study these processes owing to the technical challenges of working with a very limited amount of sample5, 6, 7. Conventional DNA methylation analysis methods typically require large amounts of starting material. Furthermore, pooling of material to achieve critical mass for conventional analysis may mask the epigenetic heterogeneity that is otherwise found in these complex mixtures of cells1.Although genome-scale DNA methylation profiling has u