CIPHER-seq enables intracellular multimodal profiling of cytokine responses in single immune cells

Abstract Single-cell RNA sequencing inadequately predicts protein abundance, particularly for intracellular cytokines. We developed CIPHER-seq (Cytokine Intracellular Protein High-throughput Expression with RNA-sequencing), enabling simultaneous quantification of intracellular proteins and transcriptomes. This system provides five layers of analysis. CIPHER-seq matched commercial protocols in immune cell recovery while significantly reducing mitochondrial stress signatures. Applied to stimulated PBMCs, CIPHER-seq resolved robust cytokine induction and metabolic remodeling, establishing a streamlined platform for intracellular multimodal profiling.