使用MAPit同时对内源性C-5 DNA甲基化和染色质可及性进行单分子检测。

Simultaneous single-molecule detection of endogenous C-5 DNA methylation and chromatin accessibility using MAPit.

作者信息

Darst Russell P, Pardo Carolina E, Pondugula Santhi, Gangaraju Vamsi K, Nabilsi Nancy H, Bartholomew Blaine, Kladde Michael P

机构信息

Department of Biochemistry and Molecular Biology, University of Florida and Shands Cancer Center, University of Florida College of Medicine, Gainesville, FL, USA.

出版信息

Methods Mol Biol. 2012;833:125-41. doi: 10.1007/978-1-61779-477-3_9.

DOI:10.1007/978-1-61779-477-3_9

PMID:22183592

Abstract

Bisulfite genomic sequencing provides a single-molecule view of cytosine methylation states. After deamination, each cloned molecule contains a record of methylation within its sequence. The full power of this technique is harnessed by treating nuclei with an exogenous DNMT prior to DNA extraction. This exogenous methylation marks regions of accessibility and footprints nucleosomes, as well as other DNA-binding proteins. Thus, each cloned molecule records not only the endogenous methylation present (at CG sites, in mammals), but also the exogenous (GC, when using the Chlorella virus protein M.CviPI). We term this technique MAPit, methylation accessibility protocol for individual templates.

摘要

亚硫酸氢盐基因组测序提供了胞嘧啶甲基化状态的单分子视图。脱氨后，每个克隆分子在其序列中都包含甲基化记录。在DNA提取之前，通过用外源DNA甲基转移酶处理细胞核来充分利用该技术的全部功能。这种外源甲基化标记了可及性区域，并确定了核小体以及其他DNA结合蛋白的足迹。因此，每个克隆分子不仅记录了（哺乳动物中CG位点的）内源性甲基化，还记录了外源性甲基化（使用小球藻病毒蛋白M.CviPI时为GC）。我们将此技术称为MAPit，即单个模板的甲基化可及性方案。