GLASS-seq: a gel-anchored, ligation-assisted, scalable biosensing platform for low-cost regional spatial transcriptomics

Spatial transcriptomics remains limited by cost, equipment burden, and inefficient detection of predefined low-abundance targets in small regions of interest (ROIs). We present GLASS-seq (Gel-anchored, Ligation-Assisted, Scalable Spatial sequencing), a hydrogel-embedded, probe-ligation biosensing platform that converts in situ RNA recognition into digital sequencing readouts at ∼100 μm lateral resolution without specialized instrumentation. Target-specific split probes hybridize and are joined by SplintR ligase only upon correct dual recognition; the ligation products are reversibly immobilized within a polyacrylamide network via acrylamide-tagged anchors to limit diffusion through staining, imaging, and microdissection. A minimal PCR appends spatial X/Y barcodes to generate ROI-indexed libraries. Across mouse tissues, GLASS-seq achieved high capture fidelity (>96% correct probe-pair recovery), robust read retention to correct sequences (≈81%), strong reproducibility (r ≥ 0.98), with high signal-to-background against stringent negatives (-ligase, -anchor, single-arm, mismatch). In brain sections, a 731-gene panel profiled 212 grid-sampled ROIs at 500 μm spacing using operator-guided grid dissection, yielding contiguous domains aligned with gross neuroanatomy and strong concordance with public ISH atlases (r = 0.85) under modest sequencing (∼0.42 Gb per ROI) and a representative per-section cost of ∼$512. The workflow is compatible with immunofluorescence and in situ hybridization, and generalizes to heart, liver, spleen, lung, and kidney. Together, by framing in situ ligation and hydrogel anchoring as an arrayed molecular biosensor with sequencing as the digital transducer, GLASS-seq provides a robust, economical, and scalable approach for regional spatial RNA quantification suited for large cohorts and translational use.