UC Irvine

The architecture of cells and the tissue they form within multicellular organisms are highly complex and dynamic. Cells optimize their function within tissue microenvironments by expressing specific subsets of RNAs. Advances in cell tagging methods enable spatial understanding of RNA expression when merged with transcriptomics. However, these techniques are currently limited by the spatial resolution of the tagging, the number of RNAs that can be sequenced, and multiplexing to isolate spatially-distinct cells within the same tissue landscape. To address these limitations, we developed CrossSeq, which employs photocrosslinking fluorescent probes and confocal microscopy activation to demarcate user-defined regions of interest on fixed cells for multiplexed spatial transcriptomic analysis. We investigate phenyl azide and diazirine crosslinking scaffolds and define their photoactivity profiles. We then deploy the aryl azide scaffold with three fluorophores for multiplexing on glyoxal fixed cells and analyze the defined populations using flow cytometry. Finally, we apply CrossSeq to investigate an in vitro MDA-MB-231-LM2 metastatic cancer migration model to evaluate changes in gene expression at the migratory cell front versus the exterior population. We anticipate this new technology will be a valuable tool addition as it will enable easier access to spatial transcriptomic analysis for the scientific community using conventional microscopy and analysis techniques.