Advanced eTAM-seq enables high-fidelity, low-input N ⁶ -methyladenosine profiling in human cells and embryonic mouse tissues

Functional dissection of N6-methyladenosine (m ⁶ A), the most abundant internal messenger RNA modification in mammals, demands quantitative, scalable detection technology. We previously reported eTAM-seq, which supports transcriptome-wide quantification of m ⁶ A by enzyme-assisted adenosine deamination. While effective, TadA8.20—the enzyme used in our first-generation technology—is sensitive not only to m ⁶ A but also to RNA structure, making accurate detection dependent on the inclusion of control transcriptomes. Here, we introduce eTAM-seq-v2, in which we replace TadA8.20 with TadA8r, a further evolved adenosine deaminase with superior catalytic efficiency. eTAM-seq-v2 supports control-free m ⁶ A calling with high fidelity. Because enzyme treatment preserves RNA integrity, eTAM-seq surveys >51% of A sites in all expressed genes with moderate sequencing depth (60 million uniquely mapped reads) and delivers robust performance with as little as 10 ng of total RNA (∼500 cells). With eTAM-seq-v2, we delineate the m ⁶ A landscape across six human cell lines and seven embryonic mouse tissues. While uncovering broadly conserved m ⁶ A patterns, we reveal that most neighboring m ⁶ A sites are independently deposited at the single-molecule level. Moving forward, we envision that eTAM-seq-v2 will enable researchers to survey m ⁶ A in diverse biological contexts and uncover new insights into its regulatory roles.