A protocol for high-quality single-cell RNA sequencing with cell surface protein quantification.

Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) enables the simultaneous analysis of transcriptomic and proteomic data at the single-cell level, providing a comprehensive view of cellular heterogeneity and function. In this study, we present a standardized approach for high-quality single-cell RNA sequencing coupled with cell surface protein quantification. Key advantages of CITE-seq include its compatibility with existing scRNA-seq workflows, cost-efficient high-throughput protein detection, and enhanced resolution in cell type classification. Detailed steps for sample preparation, antibody-oligo conjugation, gel bead-in-emulsion (GEM) generation, complementary deoxyribonucleic acid (cDNA) amplification, and library construction are provided, ensuring reproducibility and robust data quality. This protocol facilitates the integration of multimodal single-cell data, enabling precise characterization of rare cell subsets and advancing insights in immunology, oncology, and developmental biology. The workflow is optimized for flexibility across platforms and scalable for diverse research applications.