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

Area of Science:

• Single-cell multi-omics analysis
• Molecular biology techniques
• Biotechnology applications

Background:

• Single-cell analysis reveals cellular heterogeneity.
• Simultaneous transcriptomic and proteomic data provide deeper biological insights.
• Existing methods may lack comprehensive cell surface protein information.

Purpose of the Study:

• To present a standardized protocol for CITE-seq.
• To enable high-quality single-cell RNA sequencing coupled with cell surface protein quantification.
• To facilitate integration of multimodal single-cell data.

Main Methods:

• Detailed protocol for sample preparation and antibody-oligo conjugation.
• Gel bead-in-emulsion (GEM) generation and cDNA amplification.
• Library construction for high-throughput sequencing and protein detection.

Main Results:

• Demonstrated compatibility with existing scRNA-seq workflows.
• Achieved cost-efficient, high-throughput protein detection.
• Enhanced resolution in cell type classification and identification of rare cell subsets.

Conclusions:

• The standardized CITE-seq protocol ensures reproducibility and robust data quality.
• This workflow advances insights in immunology, oncology, and developmental biology.
• The protocol is flexible, scalable, and applicable across diverse research settings.