Abstract
Background: DNA methylation and chromatin accessibility are pivotal epigenetic regulators of gene expression and cellular identity, with significant implications in tumorigenesis and progression. Current single-cell multi-omics methods are limited in throughput and sensitivity, hindering comprehensive biomarker discovery. Methods: We developed single-cell split-pool ligation-based multi-omics sequencing technology (SpliCOOL-seq), a high-throughput single-cell sequencing technology that simultaneously profiles whole-genome DNA methylation and chromatin accessibility in thousands of cells. By integrating in situ GpC methylation, universal Tn5 tagmentation, and split-pool combinatorial barcoding, SpliCOOL-seq achieves enhanced sensitivity and scalability. Results: SpliCOOL-seq accurately distinguished lung cancer cell types based on genetic and multiple epigenetic modalities and revealed that the two DNA methyltransferase (DNMT) inhibitors, 5-Azacitidine and Decitabine, both cause large-scale demethylation but in distinct patterns. Applied to primary lung adenocarcinoma, SpliCOOL-seq identified tumour subclones within the tumour lesion and uncovered novel DNA methylation biomarkers (e.g., FAM124B, SFN, OR7E47P) associated with patient survival. Additionally, we demonstrated accelerated epigenetic ageing and mitotic activity in tumour subclones, providing new insights into tumorigenesis. Conclusion: SpliCOOL-seq achieves parallel profiling of whole-genome DNA methylation and chromatin accessibility in the same individual cells in a high-throughput manner and is hopefully used to illustrate regulatory interactions under different cell states. SpliCOOL-seq enables high-resolution, multi-modal epigenetic profiling at single-cell resolution, offering a powerful platform for discovering cancer biomarkers. Its application reveals novel therapeutic targets and early-diagnostic markers, underscoring its potential in precision oncology. Key points: SpliCOOL-seq achieves high-throughput single-cell co-profiling of DNA methylation and chromatin accessibility. DNMT inhibitors caused cancer cell demethylation with divergent patterns. SpliCOOL-seq enables the discovery of genes related to LUAD tumorigenesis. Ageing and LUAD tumorigenesis may share similar epigenetic alterations.