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

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. Highlights DISCO-seq integrates RNA-preserving tissue-clearing chemistry with whole-organ or organism 3D imaging, enabling anatomically unbiased single-cell transcriptomics. DISCO-seq yields RNA quality and transcriptome profiles equivalent to those obtained from matched fresh or fixed tissues. DISCO-seq identifies discrete glioblastoma microenvironments and defines their transcriptomic states within the intact brain. DISCO-seq enables whole-body mapping of SARS-CoV-2 S1 and uncovers region-specific immune and metabolic responses across anatomical niches. HighlightsSupplementary movies can be seen at: http://discotechnologies.org/DISCO-seq/ Competing Interest Statement A.E. is co-founder of Deep Piction, GmbH. M.B. is a member of the Neuroimaging Committee of the EANM. M.B. has received speaker honoraria from Roche, GE Healthcare, Iba, and Life Molecular Imaging; has advised Life Molecular Imaging, AC Immune, MIAC, and GE healthcare.