University College London (UCL) Cancer Institute, University College London, London, UK.
Nat Protoc. 2012 Mar 8;7(4):617-36. doi: 10.1038/nprot.2012.012.
DNA methylation is an epigenetic mark that has a crucial role in many biological processes. To understand the functional consequences of DNA methylation on phenotypic plasticity, a genome-wide analysis should be embraced. This in turn requires a technique that balances accuracy, genome coverage, resolution and cost, yet is low in DNA input in order to minimize the drain on precious samples. Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) fulfils these criteria, combining MeDIP with massively parallel DNA sequencing. Here we report an improved protocol using 100-fold less genomic DNA than that commonly used. We show comparable results for specificity (>97%) and enrichment (>100-fold) over a wide range of DNA concentrations (5,000-50 ng) and demonstrate the utility of the protocol for the generation of methylomes from rare bone marrow cells using 160-300 ng of starting DNA. The protocol described here, i.e., DNA extraction to generation of MeDIP-seq library, can be completed within 3-5 d.
DNA 甲基化是一种表观遗传标记，在许多生物学过程中起着关键作用。为了了解 DNA 甲基化对表型可塑性的功能后果，应该进行全基因组分析。这反过来又需要一种既能平衡准确性、基因组覆盖度、分辨率和成本，又能在 DNA 输入量低的技术，以尽量减少珍贵样本的消耗。甲基化 DNA 免疫沉淀测序（MeDIP-seq）满足这些标准，它将 MeDIP 与大规模平行 DNA 测序相结合。在这里，我们报告了一个改进的方案，该方案使用的基因组 DNA 量比常用的方案少 100 倍。我们在广泛的 DNA 浓度（5,000-50ng）范围内显示了特异性（>97%）和富集（>100 倍）的可比性结果，并证明了该方案在使用 160-300ng 起始 DNA 从稀有骨髓细胞生成甲基组方面的实用性。本文描述的方案，即从 DNA 提取到 MeDIP-seq 文库的生成，可在 3-5 天内完成。
