Background While scRNA-seq has transformed our view of cellular diversity, it misses protein expression, crucial for cell function. InTraSeq (Intracellular protein and Transcriptomic Sequencing), a novel technology by Cell Signaling Technology, overcomes that limitation.

Methods InTraSeq simultaneously measures RNA, surface markers, and proteins across compartments within single cells, using antibodies for in-depth analysis. Validated in the 10x Genomics workflows, it requires minimal handling and allows multi-day storage, ideal for high-throughput studies.

Results Our study confirms InTraSeq’s effectiveness in preserving RNA and cellular heterogeneity. Protein signals from InTraSeq antibodies generally aligned with RNA expression, but discrepancies emerged, especially for post-translational modifications (PTMs). Notably, InTraSeq distinguished T cell states undetectable by scRNA-seq alone.

Drug treatment experiments using InTraSeq revealed expected changes in signaling pathways, mirroring findings from T cell stimulation and treatment. Analysis of Th17 cell differentiation showcased dynamic shifts in RNA, protein levels, and PTMs, confirming early Stat3 protein activation. These results highlight InTraSeq’s potential to elucidate signaling pathways and uncover novel biological insights.

Conclusions InTraSeq emerges as a powerful tool for dissecting cellular complexity at the molecular level. Its ability to identify correlations between protein expression, PTMs, and RNA profiles within specific cell types of complex tissues holds immense promise. InTraSeq facilitates discovery of novel cell subtypes, mapping of signaling pathways, and understanding of cell differentiation trajectories. This comprehensive approach has the potential to revolutionize our understanding of cell biology by unveiling cellular mechanisms hidden in scRNA-seq data. Analyzing Th17 differentiation dynamics exemplifies the exciting possibilities offered by InTraSeq.