Multiplexed quantification of proteins and transcripts in single cells

Area of Science:

• Single-cell biology
• Molecular biology
• Immunology

Background:

• Accurate measurement of gene and protein expression is crucial for understanding cellular function.
• Existing methods often require separate workflows for gene and protein analysis, limiting throughput and comprehensive analysis.
• Single-cell resolution is key to dissecting cellular heterogeneity and identifying rare cell populations.

Purpose of the Study:

• To introduce a novel assay, RNA expression and protein sequencing assay (REAP-seq), for simultaneous quantification of gene and protein expression in single cells.
• To demonstrate the utility of REAP-seq in a biological context, including assessing drug effects and discovering novel cell types.

Main Methods:

• Development and application of REAP-seq, integrating DNA-labeled antibodies and droplet microfluidics.
• Quantification of >20,000 genes and proteins using 82 barcoded antibodies in a single workflow.
• Application of REAP-seq to analyze human CD8+ lymphocytes and investigate the effects of a CD27 agonist.

Main Results:

• Successful simultaneous measurement of gene and protein expression at the single-cell level.
• Characterization of the costimulatory effects of a CD27 agonist on human CD8+ lymphocytes.
• Identification and characterization of a previously unknown cell type within the analyzed samples.

Conclusions:

• REAP-seq provides a powerful, integrated approach for high-throughput single-cell multi-omics analysis.
• This assay enables deeper insights into cellular responses and facilitates the discovery of novel cellular identities.
• REAP-seq has broad applications in immunology, cancer research, and developmental biology.