Spatial DNA-directed single-molecule methylome and transcriptome co-profiling in mammalian tissues

DNA cytosine modifications, including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), are fundamental epigenetic marks that regulate and maintain cell-type-specific transcriptional programs. 1 Recent advances in single-cell and spatial methylome profiling have enabled characterization of epigenetic heterogeneity across cell populations and developmental contexts. 2 , 3 , 4 However, existing methylome profiling strategies lack the spatial resolution needed to simultaneously capture 5mC and 5hmC landscapes within tissue microenvironments. Third-generation sequencing technologies, particularly Oxford Nanopore sequencing, enable direct detection of 5mC and 5hmC without bisulfite or enzymatic conversion. 5 This approach preserves DNA integrity, avoids amplification bias introduced by polymerase chain reaction (PCR), and generates long reads that support haplotype-resolved epigenomic analysis. 5 Here, we developed Spatial DNA-Directed Single-Molecule Methylome & High-Throughput Transcriptome Sequencing (Spatial-DiSMHT-seq), a spatial multi-omics technology that enables joint profiling of native 5mC and 5hmC landscapes and transcriptomes in intact tissues.