A multi-modal single-nucleus atlas of diverse tissues and cell lines via DUET-seq
Jointly profiling chromatin accessibility and gene expression in the same single cell is a powerful approach for linking regulatory elements to transcriptional output, yet widespread adoption has been limited by poor co-capture sensitivity, proprietary closed-source platforms, and prohibitive costs. Here we present DUET-seq, an open-source droplet microfluidic platform that jointly profiles chromatin accessibility and gene expression from the same nucleus. DUET-seq introduces programmable, dissolvable dual-linker hydrogel beads and a one-step intra-droplet RT-PCR chemistry that physically co-indexes RNA and transposed chromatin fragments, completing library preparation in under 12 hours. Systematic optimization of joint reaction conditions, including strategies to suppress residual Tn5 activity, yields transcriptomic sensitivity exceeding that of existing platforms (approximately 3,200 genes per cell in HEK293T) while maintaining competitive chromatin signal quality. By providing a fully disclosed reagent-and-device ecosystem, DUET-seq enables researchers to optimize lysis and reaction chemistry for non-standard tissues without proprietary constraints. We demonstrate the platform's versatility by mapping over 12,000 cis-regulatory element-to-gene linkages in adult mouse brain and by profiling approximately 29,000 nuclei across six stages of postnatal spermatogenesis, where paired measurements reveal two modes of temporal regulatory decoupling - epigenetic priming and chromatin inertia - that are inaccessible to unimodal assays. DUET-seq thus provides an accessible, cost-effective framework for joint single-nucleus multi-omic profiling, with broad applicability across developmental biology, disease epigenomics, and functional genomics.