LEAD-m6A-seq for Locus-specific Detection of N6-methyladenosine and Quantification of Differential Methylation.,Angewandte Chemie International Edition

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LEAD-m6A-seq for Locus-specific Detection of N6-methyladenosine and Quantification of Differential Methylation.
Angewandte Chemie International Edition ( IF 16.1 ) Pub Date : 2020-09-24 , DOI: 10.1002/anie.202007266
Yuru Wang _{1,

2} , Zijie Zhang _{1,

2,

3,

4} , Caraline Sepich-Poore _{1,

2,

3,

5} , Lisheng Zhang _{2,

4} , Yu Xiao ₂ , Chuan He _{1,

2,

3,

4}

Affiliation

N⁶‐methyladenosine (m⁶A) is a crucial RNA chemical mark which plays important roles in various biological processes. The development of highly multiplexed, cost‐effective, and easy‐to‐operate methodologies for locus‐specific analysis of m⁶A is critical for advancing our understanding of the roles of this modification. Herein, we report a method which builds upon the principle of the previously reported SELECT approach by significantly improving its efficiency and coupling it to next generation sequencing technology for high‐throughput validation and detection of m⁶A modification at selected sites (LEAD‐m⁶A‐seq). Through probing cDNA extension mediated by Bst DNA polymerase at and near target cellular sites by sequencing, we evaluated m⁶A modification at these sites, and estimated differential methylation levels (0–84 %) upon in vitro demethylation by the m⁶A demethylase FTO with high reproducibility. We envision that this strategy can be readily used for testing a greater number of sites with a broad dynamic range and modified to study other RNA modifications.

中文翻译：

LEAD-m6A-seq 用于 N6-甲基腺苷的位点特异性检测和差异甲基化的定量。

N ⁶ -甲基腺苷（m ⁶ A）是一种重要的RNA化学标记，在各种生物过程中发挥着重要作用。开发高度多重、经济高效且易于操作的 m ⁶ A 位点特异性分析方法对于加深我们对这种修饰作用的理解至关重要。在此，我们报告了一种基于先前报道的 SELECT 方法原理的方法，显着提高了其效率并将其与下一代测序技术相结合，以进行高通量验证和检测选定位点的 m ⁶ A 修饰（LEAD-m ⁶ A-seq）。通过测序探测 Bst DNA 聚合酶在靶细胞位点及其附近介导的 cDNA 延伸，我们评估了这些位点的 m ⁶ A 修饰，并估计了 m ⁶ A 去甲基化酶 FTO 体外去甲基化后的差异甲基化水平 (0–84 %)具有高重现性。我们设想这种策略可以很容易地用于测试更多具有广泛动态范围的位点，并进行修改以研究其他 RNA 修饰。

更新日期：2020-09-24

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