Identification of Methylated Deoxyadenosines in Genomic DNA by dA6m DNA Immunoprecipitation.
Identification of Methylated Deoxyadenosines in Genomic DNA by dA6m DNA Immunoprecipitation.
复制标题
DOI：
10.21769/bioprotoc.1990
复制
发表时间：
2016-11
期刊：
影响因子：
0.8
通讯作者：
Magdalena J. Koziol;C. Bradshaw;George E. Allen;Ana S. H. Costa;C. Frezza
中科院分区：
文献类型：
--
作者：
Magdalena J. Koziol;C. Bradshaw;George E. Allen;Ana S. H. Costa;C. Frezza
dA 6 m DNA免疫沉淀后进行深度测序（DIP-Seq）是鉴定和研究N6-甲基脱氧腺苷（dA 6 m）全基因组分布的关键工具。这种新的DNA修饰的精确功能仍有待充分阐明，但已知其不存在于转录起始位点并被排除在外显子之外，表明其在转录调控中的作用（Koziol et al.，2015年）。重要的是，它的存在表明DNA可能比以前认为的更多样，因为在真核DNA中可能存在进一步的DNA修饰（Koziol et al.，2015年）的报告。该方案描述了进行dA 6 m DNA免疫沉淀（DIP）的方法，如应用于表征高等真核生物中的第一个dA 6 m甲基化组分析（Koziol et al.，2015年）。在该方案中，我们描述了如何分离基因组DNA，片段化，然后用识别基因组DNA中dA 6 m的抗体将含有dA 6 m的DNA拉下。在随后的洗涤后，除去不含dA 6 m的DNA片段，并从抗体中洗脱含dA 6 m的片段，以便进一步处理用于随后的分析。开发该方案是为了鉴定基因组中含有dA 6 m的区域。它可以用于检测不同基因组中的dA 6 m。作为指导，该方案是根据用于检测RNA中腺苷甲基化的现有方法建立的（Dominissini等人，2013年）的报告。我们开发了这种方案，并将其用于检测DNA中的dA 6 m，而不是检测腺苷甲基化RNA。这是必需的，因为当时没有协议可以允许在真核DNA中全基因组鉴定dA 6 m。
dA6m DNA immunoprecipitation followed by deep sequencing (DIP-Seq) is a key tool in identifying and studying the genome-wide distribution of N6-methyldeoxyadenosine (dA6m). The precise function of this novel DNA modification remains to be fully elucidated, but it is known to be absent from transcriptional start sites and excluded from exons, suggesting a role in transcriptional regulation (Koziol et al., 2015). Importantly, its existence suggests that DNA might be more diverse than previously believed, as further DNA modifications might exist in eukaryotic DNA (Koziol et al., 2015). This protocol describes the method to perform dA6m DNA immunoprecipitation (DIP), as was applied to characterize the first dA6m methylome analysis in higher eukaryotes (Koziol et al., 2015). In this protocol, we describe how genomic DNA is isolated, fragmented and then DNA containing dA6m is pulled down with an antibody that recognizes dA6m in genomic DNA. After subsequent washes, DNA fragments that do not contain dA6m are eliminated, and the dA6m containing fragments are eluted from the antibody in order to be processed further for subsequent analyses. BACKGROUND This protocol was developed in order to identify regions in the genome that contain dA6m. It can be used to detect dA6m in different genomes. As a guideline, this protocol was established from existing approaches used to detect adenosine methylation in RNA (Dominissini et al., 2013). We developed this protocol and adapted it for the detection of dA6m in DNA, rather than detecting adenosine methylation RNA. This was required, as no protocol was available at that time to allow the genome-wide identification of dA6m in eukaryotic DNA.