Well-ST-seq: Cost-Effective and Near-Cellular Spatial Transcriptomics Using Deterministic Barcoded Bead Arrays.

Spatial transcriptomic technologies are promising tools for elucidating fine anatomical profiles of tissues. For methods that rely on deterministic probe arrays, balancing spatial resolution, cost, and transcript-capture sensitivity is crucial to advancing spatial transcriptomics in both basic research and clinical applications. Here we present Well-ST-seq, a near-cellular-resolution platform that integrates microwell-assembled hydrogel bead carriers with combinatorial microfluidic indexing to generate predefined spatial barcode arrays. In this design, orthogonal microchannels are used for coordinate indexing, while probe construction is confined to bead carriers and executed through a simplified workflow, minimizing the need for biochemical processing inside narrow channels. Consequently, spatially barcoded capture arrays can be prepared in ∼2 h at a direct consumables cost of ∼$0.05-$0.54 per mm2 for 30-10 μm spot sizes. Using 10 μm arrays, Well-ST-seq achieves high transcript recovery in mouse hippocampus, yielding 3,896 UMIs per spot, and supports the delineation of layered organization and region-specific expression programs. Across consecutive sections of developing mouse embryonic brain, our method further enables coherent alignment and consistent domain-level mapping throughout the series. Together, Well-ST-seq provides a promising route to rapid, cost-efficient fabrication of deterministic spatial transcriptomics slides and to near-cellular spatial tissue profiling.