An integrated single-cell multiomics approach to characterize mRNA, intracellular, and surface proteins using intracellular AbSeq and BD (R) AbSeq Immune Discovery Panel

Abstract DNA-barcoded antibodies enable single-cell multiomics approaches that examine protein alongside mRNA in a single cell. Although it is a powerful approach, leading methods are currently limited to surface proteins, which limits understanding of signal-transduction and transcriptional pathways that are often governed by expression changes and posttranslational modifications of intracellular proteins. The ability to analyze intracellular proteins alongside the transcriptome is complicated by the fix and perm processes necessary to access intracellular epitopes, which can negatively impact RNA assay sensitivity. Furthermore, nonspecific binding of antibody-oligos to nucleic acids can generate high background signals. To address these issues, we generated a protocol that can recover high-quality RNA alongside specific intracellular and surface protein information. Using this protocol in conjunction with the BD Rhapsody™ Single-Cell Analysis System, we simultaneously profiled over 50 protein markers including 14 intracellular proteins with the whole transcriptome. We identified predicted patterns, including increased phosphorylation of AKT protein correlated with the proliferation marker Ki67 upon immune stimulation, and transcription factor (T-bet) expression in a specific cell subset. Together these preliminary results suggest that analysis of intracellular proteins together with the transcriptome may be possible using single-cell sequencing technologies and can enable a deeper understanding of cellular states. For Research Use Only. Not for use in diagnostic or therapeutic procedures.