Highly sensitive, sub-micron spatial host-microbiome transcriptomics in FFPE tissues via iterative hydrogel expansion

Here, Ex-spRandom is presented, a highly sensitive platform that integrates random-primed in situ cDNA synthesis with an interpenetrating polymer network (IPN)-based tissue expansion protocol.

This physicochemical synergy physically decrowds the dense FFPE matrix, directly converting fragmented host and microbial RNAs into stably anchored cDNA. Consequently, Ex-spRandom translates macro-scale arrays into a sub-micron effective resolution of ~0.9 µm while achieving an unprecedented transcriptomic sensitivity exceeding 40,000 unique genes per sample. This platform successfully rescues non-coding regulatory transcripts and unmasks continuous neurodevelopmental trajectories in the dense embryonic eye.