Background: The tumor microenvironment is a complex ecosystem where the spatial relationships between cells influence gene and protein expression. To understand disease progression, it is essential to correlate molecular expression with tissue context and cellular interactions. Direct in-sample RNA sequencing (DiSS) enables untargeted transcriptome quantification by sequencing RNA molecules in their native tissue environment. Here, we combined DiSS chemistry with Teton™ protein detection to deliver a multimodal RNA and protein readout sampled directly from the cells and regions where they are expressed.

Untargeted transcriptome detection is achieved via poly-T probes that hybridize to polyadenylated mRNA, followed by primer extension, probe circularization, and rolling-circle amplification for in situ sequencing. The platform directly sequences the transcriptome rather than employing probe detection via barcode counting. Using AVITI24™ sequencing chemistry, we obtained high-fidelity transcriptome data across a range of sample types. The platform’s enhanced read quality is capable of high-quality base calling through difficult homopolymer-rich regions common in RNA to support accurate base calling in both fresh frozen and FFPE tissue. Using AVITI24, we were able to investigate a multitude of tissue conformations because of the 20 cm² imageable area per dual sided run, exploring large tissue sections, consecutive sections across the z-plane, and tissue microarrays. Our workflow enabled robust transcriptome and protein detection in various tissue types, including brain, liver, and lung, requiring minimal hands-on time and while preserving sample integrity for downstream assays such as H&E staining.

In both healthy and diseased tissue, we generated a high-resolution spatial map of 3′ transcript expressions and multiplexed protein profiles in healthy and diseased tissue. These maps revealed distinct gene expression patterns and cell subtypes aligned with histological architecture, highlighting functional organization within the tissue.

These results demonstrated have proven DiSS chemistry to be compatible for biological analysis across a range of sample types on the system. From prepared libraries to cells, and now tissue, this application highlights the comprehensive utility of this single platform.