Dissection of single-cell gene regulation by simultaneous digital mRNA and protein quantification

Autor: Regina Lam, Eleen Shum, Christina Christina Chang, Gretchen Lam, Nidhanjali Bansal, Hemi Shah, Devon Jensen, Jing Hu, James Ghadiali, Jody Martin, David Rosenfeld, H Christina Fan
Rok vydání: 2018
Předmět:
Zdroj: The Journal of Immunology. 200:120.41-120.41
ISSN: 1550-6606
0022-1767
DOI: 10.4049/jimmunol.200.supp.120.41
Popis: 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 – have thousands of protein copies per cell, yet is fueled by a small number of mRNA transcripts. To bridge the understanding of protein and mRNA expression differences, we used BD™ AbSeq on the Rhapsody™ 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 AbSeq 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.
Databáze: OpenAIRE
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