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

Abstract

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.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

* We re-analyzed all data considering color blindness and verified our findings with separate datasets, enhancing the feasibility of our research. We also demonstrated the feasibility of DNA methylome by whole genome bisulfite sequencing from single-nucleus samples. We added centrality-focused network analysis to visualize the connection of subcellular components.