Dissection Of Single-Cell Transcriptional And Translational Regulation By Digital Mrna And Protein Quantification

The immune system consists of complex gene regulatory networks that allow a rapid transition of different cellular states during an immune response. Cell-surface marker analysis using flow cytometry or single cell RNA-seq has allowed characterization of immune subpopulations and a greater understanding of the complexity of immune cells. However, restrictions on protein-only or RNA-only analysis can greatly limit the understanding of how genes are regulated in cells. For example, many cell surface markers - such as CD4 in T cells - has thousands of protein copies per cell using antigen density calculations, yet is fueled by a small number of mRNA transcripts per CD4+ T cell. Moreover, conventional whole-transcriptome analysis of mRNA can further mute the expression detection of CD4 mRNA in T cells due to the abundance of housekeeping ribosomal genes. To bridge the understanding of protein and mRNA expression differences, we used Ab-Seq on BD Rhapsody TM platform to provide digital quantification of both protein and mRNA expression level in single cells. An oligo-conjugated antibody panel against immune-relevant cell-surface markers was created and used for this multi-omic gene expression profiling effort. This approach is coupled with mRNA analysis using the BD Rhapsody Immune Response Panel, a targeted method of RNA-seq that allows a higher sensitivity of immune markers than conventional whole transcriptome assays. We studied the dynamics of early T cell activation in vitro to understand this response on transcriptional, post-transcriptional, and translational levels. Different time points following anti-CD3 and anti-CD28 treatment were collected and multiplexed on to BD Rhapsody cartridge for single cell capture and analysis. Using Ab-Seq on BD Rhapsody, we were able to detect the difference in mRNA and protein levels of crucial markers, allowing us to dissect the intricate gene regulatory pathways during an immune response in a single cell level. Citation Format: Gretchen Lam, Eleen Shum, Christina Chang, Hemi Shah, Devon Jensen, James Ghadiali, Jody Martin, David Rosenfeld, Christina H. Fan. Dissection of single-cell transcriptional and translational regulation by digital mRNA and protein quantification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1419.