RNA <em>In situ</em> Hybridization with Sequential Protein Immunofluorescence in Tandem Assay

The functionality of cells within a host does not depend on isolated signals. Instead, all components, from the smallest RNA molecules to the largest proteins, must operate in harmony and coordination within the human body. As such, the importance of approaches that integrate cellular-level spatial investigations across multiple omics is paramount to understanding cell-cell interactions and the progression of disease. Dissecting the proteome and transcriptome in the same spatial assay helps us understand how not only what a cell is being instructed to carry out (RNA), but also how it is executing those instructions in the context of the microenvironmental niche it finds itself in (protein). This manuscript is focused on integrating sequential immunofluorescence (SeqIF) with RNA in situ hybridization (ISH) with an on-tissue microfluidics driven system for high-throughput protein and RNA investigation in a spatial context (seqRNA-ISH+seqIF) that will allow up to 12 RNA and 24 protein targets in a single run with additional protein targets possible to be added via sequential runs. This method provides a sequential targeted multiomics platform that does not require consideration of fluorophore compatibility and extensive optimization for higher plexing. This allows one to understand what messages the cell is priming or is sending into its microenvironment. This targeted approach helps to validate whole transcriptome methods while examining the interactions between the RNA and proteins in a more precise manner.