Advancing single cell technology: iSCseq drives living subcellular transcriptomic profiling in osteoimmune diversity

SUMMARY Single-cell RNA-seq (scRNA-seq) has clarified cellular heterogeneity within cell populations. However, scRNA-seq and spatial transcriptomics cannot capture the dynamic transcriptomic changes inside living cells. To decode subcellular gene expression, we developed intra-single cell sequencing (iSCseq), a novel approach that combines confocal imaging, repeatedly picking up cellular components inside living cells, and next-generation sequencing (intra single-cell RNA-seq; iSCseq). iSCseq illustrated the subcellular heterogeneity of gene expression. iSCseq revealed not only multiple differentiation stages embedded in the same cell, but also physical cytoskeletal connections, physiological activity of mitochondria, and intracellular calcium, as confirmed by transcriptomic evidence. Inclusive iSCseq with in vivo scRNA-seq datasets identified new osteoclast subsets in physiological and pathological bones. Network analysis with centrality provided insights into the connection between subcellular components, and clearly divided differentiation and fusion processes in multinucleation. The iSCseq approach has the potential to enhance cell biology at subcellular resolution and identify new therapeutic targets. Graphical abstract In brief intra-single cell sequencing (iSCseq) enhances single-cell technology by combining live cell imaging, subcellular sampling from living cells and sequencing, offering deeper insights into cell functions and pathology at subcellular resolution through inclusive analysis with scRNA-seq and advanced centrality-focused network analysis. Highlights intra-single cell sequencing (iSCseq) clarifies subcellular heterogeneity iSCseq connects morphological and physiological features with transcriptome Inclusive iSCseq unveils osteoclast subsets in physiological and pathological bones Linkage at subcellular resolution reveals key players in characteristic fusion