Kaiqiang, Y.; Zhao, X.; Wenjia, W.; Handong, W.

Spatial transcriptomics (ST) has emerged as a transformative tool for resolving the molecular heterogeneity of complex tissues within their native anatomical context. However, next-generation sequencing (NGS)-based ST platforms frequently encounter sensitivity bottlenecks arising from sub-optimal probe architectures on solid substrates. Conventional single-stranded DNA coupling methods often lead to disordered interfacial molecular conformations due to non-specific nucleobase-mediated surface tethering, which creates steric hindrance and inhibits the enzymatic efficiency of in situ library preparation. Here, we present Decomap (double-strand protected combinatorial barcoding microarray chip), a high-performance ST platform utilizing a triple-segment (dsZ-X-Y) fabrication strategy to achieve superior transcript capture efficiency. This structural optimization significantly enhances DNA ligation kinetics and subsequent polymerase-mediated extension, overcoming the fundamental limitations of traditional single-stranded coupling strategies. Decomap-seq achieved a median detection of 7,200 genes and 29,097 UMIs per 50 m-spot at a sequencing saturation of 50.1%. These results validate Decomap as a highly sensitive and robust tool for spatial transcriptomics, offering a powerful platform for advancing research in histopathology, developmental biology, and neuroscience.