Single base mapping of m6A by an antibody-independent method. Single base mapping of m6A by an antibody-independent method

N6-methyladenosine (m6A) is one of the most abundant mRNA modifications in eukaryotes, related to pivotal RNA metabolism processes. The most popular high-throughput m6A identification method relies on the commercial m6A antibody but suffers from poor reproducibility and limited resolution. Exact location of m6A site is of great vital for understanding the dynamics, functions and machinery of RNA methylation. Here, we developed a precise and high-throughput antibody-independent m6A identification method based on the m6A-sensitive RNA endoribonuclease recognizing ACA motif (m6A-sensitive RNA-Endoribonuclease–Facilitated sequencing or m6A-REF-seq). Whole-transcriptomic single base m6A map generated by m6A-REF-seq displayed a typical distribution pattern with enrichment adjacent to stop codon. Ligase-based and qPCR validation methods were used to confirm the individual m6A sites and quantify the methylation level, reinforcing the high accuracy of m6A-REF-seq. We applied m6A-REF-seq on five tissues from three mammals, showing that m6A sites were conserved and tend to gather together among species. (m6A-REF-seq had been named as Aim-seq.) Overall design: For human HEK293T cell line, three pairs of replicates with MazF treatment and negative control were provided. For the samples of human tissues (brain, liver and kidney), mouse tissues (brain, liver, heart, testis and kidney) and rat tissues (brain, liver and kidney), MazF treatment and negative control were provided.

N6-甲基腺嘌呤（m6A）是真核生物中丰度最高的mRNA修饰之一，与关键的RNA代谢过程紧密关联。当前主流的高通量m6A鉴定方法依赖商业化m6A抗体，但存在重复性欠佳、分辨率有限的弊端。精准定位m6A位点，对于阐明RNA甲基化的动态变化、生物学功能及调控机制至关重要。本研究开发了一种精准且无需抗体的高通量m6A鉴定方法，该方法基于识别ACA基序的m6A敏感型核糖核酸内切酶，即m6A敏感型核糖核酸内切酶辅助测序（m6A-sensitive RNA-Endoribonuclease–Facilitated sequencing，简称m6A-REF-seq，曾被命名为Aim-seq）。通过m6A-REF-seq构建的全转录组单碱基m6A图谱，呈现出典型的分布特征：在终止密码子附近存在富集现象。本研究采用基于连接酶的验证方法与qPCR实验，对单个m6A位点进行验证并量化甲基化水平，进一步证实了m6A-REF-seq的高精度。我们将m6A-REF-seq应用于三类哺乳动物的五种组织中，结果显示m6A位点在物种间具有保守性，且倾向于聚集分布。实验整体设计：针对人源HEK293T细胞系，设置了3组生物学重复，每组均包含MazF处理组与阴性对照；对于人源组织（大脑、肝脏、肾脏）、小鼠组织（大脑、肝脏、心脏、睾丸与肾脏）以及大鼠组织（大脑、肝脏、肾脏）的样本，均设置了MazF处理组与阴性对照。