Simultaneous Detection of Transcription Factors and RNA Using TotalSeq™ Antibodies with 10x Genomics Chromium Single Cell Flex Gene Expression 2258335

Abstract Introduction Transcription factors (TFs) are key proteins that regulate gene expression by binding specific DNA sequences. They control essential cellular processes such as cell cycle, metabolism, immune responses, and more. Characterizing TFs in defined cell types is crucial to understanding their physiology and role in homeostasis and disease. While other techniques such as intracellular flow cytometry are commonly used, single-cell sequencing offers comparable resolution while providing simultaneous transcriptome characterization and higher throughput protein detection. Methods Here, we profiled human PBMCs and multiple cell lines using the 10x Genomics Chromium Fixed RNA Profiling Kit with our TotalSeq-B Human Universal Cocktail (140 antibodies), plus 13 intracellular effector proteins, and 25 TFs. Cells were stained using our optimized TotalSeq™ Cell Surface and Nuclear Protein Staining protocol, and RNA, surface, and intracellular proteins were simultaneously profiled. Results TFs such as T-BET, FOXP3, TCF1, and THPOK, and signaling proteins like ZAP70 and ITK were reliably detected in T and NK cell clusters as expected. SPI1, NTAL, BCL11B, and SYK were enriched in B cell and monocyte clusters. OCT4, NANOG, and SOX2 were detected in the human embryonal carcinoma cell line NCCIT, and FOXA1 in the luminal epithelial-like breast cancer cell line MCF-7. RNA-protein correlation was confirmed where expected. Conclusion In summary, we demonstrate that our workflow enables sensitive and specific single-cell profiling of both RNA and nuclear proteins while preserving RNA integrity. Funding Source BioLegend Topic Categories Technological Innovations in Immunology (TECH)