Immunophenotyping by Single-Cell CITE-Seq

Single-cell transcriptomics has revolutionized our understanding of cellular heterogeneity by enabling high-resolution gene expression profiling at the individual cell level. However, traditional single-cell RNA sequencing (scRNA-seq) lacks direct protein quantification, limiting comprehensive immunophenotyping. Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) overcomes this limitation by integrating antibody-derived tag (ADT) quantification with scRNA-seq, allowing simultaneous measurement of surface protein and gene expression from the same cell. This multimodal approach enhances immune cell characterization, revealing new functional states and rare subpopulations in complex biological systems. Here, we provide a detailed protocol for performing CITE-seq, from sample preparation to sequencing and data analysis. We highlight key experimental considerations, discuss challenges related to antibody selection and batch effects, and provide troubleshooting strategies to ensure robust and reproducible results. The integration of transcriptomic and proteomic data through CITE-seq provides unparalleled insights into cellular function, with broad applications in immunology, oncology, and systems biology.