DISCO-seq: 3D single-cell transcriptomics of intact biological systems

Bhatia, H. S.; Simons, L. H. A.; Kuemmerle, L. B.; McCabe, C.; Jansen, S.; He, Z.; Ali, M.; Jeridi, D.; Todorov, M. I.; Hoeher, L.; Padmarasu, S.; Park, H. E.; Thevis, J. F.; Bartos, L. M.; Singh, I.; Ussar, S.; Gorgulu, K.; Alguel, H.; Roberts, K.; Bayraktar, O. A.; Brendel, M.; Theis, F. J.; Treutlein, B.; Regev, A.; Erturk, A.

doi:10.64898/2025.12.10.693352

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bioRxiv · 10.64898/2025.12.10.693352

DISCO-seq: 3D single-cell transcriptomics of intact biological systems

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Abstract

Single-cell transcriptomics has transformed tissue analysis, yet current methods struggle to integrate whole-tissue 3D architecture. Conventional techniques restrict molecular profiling to pre-selected 2D sections, losing systemic context and introducing anatomical bias by sampling less than 0.001% of a whole organism. To overcome these challenges, we developed DISCO-seq, a tissue-clearing chemistry that enables superior RNA accessibility compared to fresh or fixed tissues. DISCO-seq integrates whole-organ or organism 3D imaging with both untargeted and targeted transcriptomics, yielding high-quality RNA from cleared tissues comparable to standard samples. We demonstrate its versatility by investigating tumor heterogeneity in a syngeneic glioblastoma mouse model, using 3D imaging to identify spatially distinct microenvironments and characterize their unique transcriptomic signatures. Moreover, DISCO-seq enabled unbiased, whole-body mapping of SARS-CoV-2 S1 protein deposition in mice, followed by transcriptomic profiling of spatially defined niches. By bridgingmesoscale 3D imaging with single-cell transcriptomics, DISCO-seq establishes a paradigm for anatomically contextualized, hypothesis-free tissue interrogation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/693352v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@758ee5org.highwire.dtl.DTLVardef@1f862c6org.highwire.dtl.DTLVardef@1cf3b9org.highwire.dtl.DTLVardef@c5082e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDISCO-seq integrates RNA-preserving tissue-clearing chemistry with whole-organ or organism 3D imaging, enabling anatomically unbiased single-cell transcriptomics. C_LIO_LIDISCO-seq yields RNA quality and transcriptome profiles equivalent to those obtained from matched fresh or fixed tissues. C_LIO_LIDISCO-seq identifies discrete glioblastoma microenvironments and defines their transcriptomic states within the intact brain. C_LIO_LIDISCO-seq enables whole-body mapping of SARS-CoV-2 S1 and uncovers region-specific immune and metabolic responses across anatomical niches. C_LI Supplementary movies can be seen at: http://discotechnologies.org/DISCO-seq/

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Bhatia, H. S., Simons, L. H. A., Kuemmerle, L. B., McCabe, C., Jansen, S., He, Z., Ali, M., Jeridi, D., Todorov, M. I., Hoeher, L., Padmarasu, S., Park, H. E., Thevis, J. F., Bartos, L. M., Singh, I., Ussar, S., Gorgulu, K., Alguel, H., Roberts, K., Bayraktar, O. A., Brendel, M., Theis, F. J., Treutlein, B., Regev, A., Erturk, A.. 2025-12-13. DISCO-seq: 3D single-cell transcriptomics of intact biological systems. https://doi.org/10.64898/2025.12.10.693352

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