Ribosome-tethered in situ sequencing for single-cell translatome analysis

Jiang, M.; He, X.; Liu, Y.; Liu, B.; Ma, W.; Zhu, L.; Lin, C.; Zhang, Y.; Ke, R.

doi:10.64898/2026.07.27.740970

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bioRxiv · 10.64898/2026.07.27.740970

Ribosome-tethered in situ sequencing for single-cell translatome analysis

Jiang, M.·He, X.·Liu, Y.·Liu, B.·Ma, W.·Zhu, L.·Lin, C.·Zhang, Y.·Ke, R.

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Abstract

Translational regulation plays a critical role in shaping cellular states and functions, yet approaches for spatially resolved translatome profiling at single-cell resolution remain limited. Here, we develop Ribosome-tethered In Situ Sequencing (Ribo-ISS), an imaging-based spatial translatomics technology that enables high-throughput mapping of ribosome-associated mRNAs in intact tissues. Ribo-ISS integrates ribosome-dependent molecular anchoring with multiplexed in situ sequencing, allowing specific detection of translation-associated transcripts without genetic manipulation or exogenous ribosome labeling. We demonstrate that Ribo-ISS achieves high specificity and enables single-cell spatial translatome profiling in mouse brain, accurately recapitulating major cell types and their anatomical organization. Applied to a sleep deprivation model, Ribo-ISS simultaneously resolved transcriptional and translational changes, revealing extensive transcription-translation uncoupling and distinct cell-type-specific translational responses. Ribo-ISS provides a scalable framework for investigating spatially organized translational programs and expands the capability of spatial omics toward understanding gene regulation beyond transcription. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/740970v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@40da8dorg.highwire.dtl.DTLVardef@1cc76b4org.highwire.dtl.DTLVardef@8535c8org.highwire.dtl.DTLVardef@18e009d_HPS_FORMAT_FIGEXP M_FIG C_FIG Ribo-ISS integrates ribosome-dependent molecular anchoring with multiplexed in situ sequencing, enabling specific detection of ribosome-bound transcripts in intact tissues without genetic manipulation or exogenous labeling. As an imaging-based spatial translatomics technology, it resolves translatome profiling at single-cell resolution. This scalable approach reveals spatial translational programs, extending spatial omics beyond transcription.

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Jiang, M., He, X., Liu, Y., Liu, B., Ma, W., Zhu, L., Lin, C., Zhang, Y., Ke, R.. 2026-07-28. Ribosome-tethered in situ sequencing for single-cell translatome analysis. https://doi.org/10.64898/2026.07.27.740970

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