N⁶-methyladenosine (m⁶A), the most abundant internal messenger RNA modification in higher eukaryotes, serves myriad roles in regulating cellular processes. Functional dissection of m⁶A is, however, hampered in part by the lack of high-resolution and quantitative detection methods. Here we present evolved TadA-assisted N⁶-methyladenosine sequencing (eTAM-seq), an enzyme-assisted sequencing technology that detects and quantifies m⁶A by global adenosine deamination. With eTAM-seq, we analyze the transcriptome-wide distribution of m⁶A in HeLa and mouse embryonic stem cells. The enzymatic deamination route employed by eTAM-seq preserves RNA integrity, facilitating m⁶A detection from limited input samples. In addition to transcriptome-wide m⁶A profiling, we demonstrate site-specific, deep-sequencing-free m⁶A quantification with as few as ten cells, an input demand orders of magnitude lower than existing quantitative profiling methods. We envision that eTAM-seq will enable researchers to not only survey the m⁶A landscape at unprecedented resolution, but also detect m⁶A at user-specified loci with a simple workflow.

N ⁶ -甲基腺苷 (m ⁶ A) 是高等真核生物中最丰富的内部信使 RNA 修饰，在调节细胞过程中发挥着多种作用。^{然而，m 6} A的功能解剖部分由于缺乏高分辨率和定量检测方法而受到阻碍。在这里，我们展示了进化的 TadA 辅助N ⁶ -甲基腺苷测序 (eTAM-seq)，这是一种酶辅助测序技术，可通过全局腺苷脱氨来检测和定量 m ^{6 A。}通过 eTAM-seq，我们分析了HeLa 和小鼠胚胎干细胞中m ⁶ A的转录组范围分布。eTAM-seq 采用的酶脱氨途径保留了 RNA 完整性，有助于从有限的输入样本中检测 m ⁶ A。除了全转录组 m ⁶ A 分析之外，我们还演示了位点特异性、免深度测序的 m ⁶ A 定量，只需十个细胞，输入需求比现有的定量分析方法低几个数量级。我们设想 eTAM-seq 将使研究人员不仅能够以前所未有的分辨率调查 m ⁶ A 景观，而且能够通过简单的工作流程在用户指定的位点检测 m ^{6 A。}