Transcriptome-wide profiling and quantification of N6-methyladenosine by enzyme-assisted adenosine deamination [eTAM-seq]

We report evolved TadA-assisted N6-methyladenosine sequencing (eTAM-seq), an enzyme-assisted sequencing technology for quantitative, base-resolution profiling of m6A. eTAM-seq functions by global adenosine deamination, enabling detection of m6A as persistent A. We demonstrate adenosine-to-inosine (I) conversion rates up to 99% using a hyperactive TadA variant. With eTAM-seq, we profile and quantify m6A in the whole transcriptomes of HeLa cells and mouse embryonic stem cells (mESCs), with simultaneous deconvolution of the transcriptome and epitranscriptome. Further, we showcase deep sequencing-free, site-specific m6A quantification with as few as 10 cells, an input demand that is at least 4 orders of magnitude lower than existing methods. Collectively, eTAM-seq enables sensitive detection and faithful quantification of m6A with limited RNA input, representing a novel solution to deciphering the epitranscriptome. Nine HeLa wild type (3 FTO-, 3 FTO+ and 3 IVT), six mESC wild type (2 FTO-, 2 FTO+ and 2 IVT), two mESC Mettl3 cko ctrl (1 FTO- and 1 FTO+) and two mESC Mettl3 cko (1 FTO- and 1 FTO+) polyA samples were collected for high-throughput sequencing.

本研究报道了进化型TadA辅助N6-甲基腺嘌呤测序（evolved TadA-assisted N6-methyladenosine sequencing, eTAM-seq），这是一种可对N6-甲基腺嘌呤（N6-methyladenosine, m6A）进行定量、单碱基分辨率分析的酶辅助测序技术。eTAM-seq的工作原理基于全局腺嘌呤脱氨反应，可使m6A以未脱氨腺苷酸的形式被保留，从而实现其特异性检测。本研究证实，使用高活性TadA变体可实现最高99%的腺嘌呤至次黄嘌呤（adenosine-to-inosine, I）转换率。借助eTAM-seq，我们可对HeLa细胞与小鼠胚胎干细胞（mouse embryonic stem cells, mESCs）的完整转录组中的m6A进行分析与定量，并同时实现转录组与表观转录组的解卷积分析。此外，本研究展示了无需深度测序的位点特异性m6A定量方法，其起始细胞用量仅需10个，这一输入需求较现有方法至少低4个数量级。综上，eTAM-seq可在有限RNA输入量下实现m6A的高灵敏检测与精准定量，为表观转录组的解析提供了全新解决方案。本研究共收集了以下样本用于高通量测序：9份HeLa野生型样本（3份FTO阴性（FTO-）、3份FTO阳性（FTO+）及3份体外转录（in vitro transcribed, IVT）样本）、6份小鼠胚胎干细胞野生型样本（2份FTO阴性、2份FTO阳性及2份体外转录样本）、2份小鼠胚胎干细胞Mettl3条件性敲除对照样本（1份FTO阴性及1份FTO阳性）以及2份小鼠胚胎干细胞Mettl3条件性敲除样本（1份FTO阴性及1份FTO阳性）的多聚腺苷酸尾（polyA）富集RNA样本。