intra-single cell sequencing (iSCseq) spotlights transcriptomic and epigenetic heterogeneity inside multinucleated osteoclast

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 O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/506360v4_ufig1.gif" ALT="Figure 1"> View larger version (73K): org.highwire.dtl.DTLVardef@115c1d8org.highwire.dtl.DTLVardef@1695ad2org.highwire.dtl.DTLVardef@1c9d257org.highwire.dtl.DTLVardef@1107ddb_HPS_FORMAT_FIGEXP M_FIG C_FIG In briefintra-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. HighlightsO_LIintra-single cell sequencing (iSCseq) clarifies subcellular heterogeneity C_LIO_LIiSCseq connects morphological and physiological features with transcriptome C_LIO_LIInclusive iSCseq unveils osteoclast subsets in physiological and pathological bones C_LIO_LILinkage at subcellular resolution reveals key players in characteristic fusion C_LI