Improving Spatial Transcriptomics with Dual-Color Coding and Cell Painting.

Spatial transcriptomics has advanced our ability to study gene expression within the spatial context of tissues and single cells. Among the diverse methodologies available, in situ transcript detection offers subcellular spatial resolution by preserving the precise coordinates of RNA molecules. A widely used approach involves padlock probe (PLP) technology, which enables rolling circle amplification and fluorescent detection of individual transcripts. To address the limitations of conventional in situ methods in multiplexing, we recently developed the Combinatorial Dual-Color (CoDuCo) assay, enabling simultaneous detection of up to 15 distinct mRNAs in a single imaging round using only seven fluorescent dyes through unique dual-color codes. However, challenges in accurate signal-to-cell assignment persist, particularly in densely packed cell aggregates. To overcome this, we integrated a cell painting-based plasma membrane staining protocol into the CoDuCo workflow, providing clear cell boundary delineation prior to hybridization. This enhanced protocol significantly improves cell-level resolution and transcript assignment fidelity. Here, we present a stepwise and scalable protocol combining membrane staining with CoDuCo in situ hybridization, optimized for high-throughput spatial transcriptomics across diverse biological samples.