Abstract Current technologies for acquiring spatial transcript information from tissue sections rely on either RNA probes or spatial barcodes. The former methods require a priori knowledge for probeset formulation; the latter have yet to achieve single cell resolution and/or transcript capture efficiencies approaching dissociative, single-cell methods. Here, we describe a novel spatial transcriptome assay called p olony (or DNA cluster)- i nde xe d l ibrary-sequencing (PIXEL-seq). It improves upon other spatial barcoding methods by employing “continuous” polony oligos arrayed across a customized gel surface. In terms of assay performance, PIXEL-seq attains ≤ 1 µm resolution and captures &gt;1,000 unique molecular identifiers/10×10 µm 2 . In other words, this global, naive platform achieves subcellular spatial transcriptome mapping while maintaining high transcript capture efficiencies.

Continuous Polony Gels for Tissue Mapping with High Resolution and RNA Capture Efficiency