Empowering multiplexed ultra-throughout ribosome profiling with RiboWich

Ribosome profiling (RiboSeq) improved the understanding of mRNA translation, enabling the precise mapping of ribosome positioning along transcripts at single-nucleotide resolution. Although various library preparation protocols overcome the need for high input material, their technical complexity and limited efficiency hinder robust profiling, preventing them from keeping pace with other sequencing techniques and applications. To move towards high-throughput and single-cell RiboSeq technologies, we developed RiboWich (Ribosome sandWich). By directly ligating adaptors to ribosome-embedded RNA fragments, RiboWich eliminates the need for ribosome purification and size-selecting ribosome footprints and tackles two major bottlenecks, expanding RiboSeq for more advanced technologies. RiboWich offers robustness in profiling and excels in detecting upstream translons in immortalized and primary cells alike. By exploiting a dual-step multiplexing strategy, RiboWich enables the simultaneous profiling of at least 96 samples while retaining sensitivity and capturing condition-specific differences in translation. By enabling scalable, low-input translatome profiling, this advancement empowers proteogenomic approaches and AI/ML-driven data analysis to uncover regulatory dynamics, neoantigens, functional small translons, and drug-responsive signatures across diverse biological contexts and users. Altogether, RiboWich represents a straightforward, versatile, and scalable ribosome profiling conceptual platform that combines accessibility, sensitivity, and throughput potential, laying the foundation for advanced single-cell RiboSeq applications.