Single-cell RNA sequencing (scRNA-seq) has revolutionized immunology by enabling high-resolution profiling of cellular heterogeneity. However, transcriptomes alone inadequately predict immune function due to extensive post-transcriptional regulation, particularly for cytokines and activation markers. Existing multimodal technologies measure surface proteins but cannot access intracellular compartments without compromising RNA quality.
This thesis presents CIPHER-seq (Cytokine Intracellular Protein High-throughput Expression with RNA-sequencing), a stress-minimized platform for simultaneous measurement of whole transcriptomes, surface markers, and intracellular proteins in single cells. CIPHER-seq preserves RNA integrity while enabling detection of 42 intracellular proteins. Application to T cell activation reveals pervasive RNA-protein discordance and temporally ordered cytokine expression dynamics. CIPHER-seq addresses a fundamental technical barrier in single-cell immunology and enables high-fidelity profiling of immune activation states.