Single-nucleus multiomic mapping of m⁶A methylomes and transcriptomes in native populations of cells with sn-m6A-CT

N⁶-methyladenosine (m⁶A) RNA modification plays important roles in the governance of gene expression and is temporally regulated in different cell states. In contrast to global m⁶A profiling in bulk sequencing, single-cell technologies for analyzing m⁶A heterogeneity are not extensively established. Here, we developed single-nucleus m6A-CUT&Tag (sn-m6A-CT) for simultaneous profiling of m⁶A methylomes and transcriptomes within a single nucleus using mouse embryonic stem cells (mESCs). m6A-CT is capable of enriching m⁶A-marked RNA molecules in situ, without isolating RNAs from cells. We adapted m6A-CT to the droplet-based single-cell omics platform and demonstrated high-throughput performance in analyzing nuclei isolated from thousands of cells from various cell types. We show that sn-m6A-CT profiling is sufficient to determine cell identity and allows the generation of cell-type-specific m⁶A methylome landscapes from heterogeneous populations. These indicate that sn-m6A-CT provides additional dimensions to multimodal datasets and insights into epitranscriptomic landscape in defining cell fate identity and states.