The story of an individual cell extends to much more than its RNA. A combination of transcriptomic and phenotypic data at the level of individual cells can fuel critical discoveries. The relative abundance of proteins and mRNA is not one-to-one. Flow cytometry has been a reliable method for studying the expression of surface proteins, but it has had limitations in terms of the number of targets that can be analyzed and its inability to provide transcriptomic data simultaneously.

CITE-seq is a new technique that can achieve both goals in a single assay, providing a comprehensive understanding of single-cell function.CITE-Seq integrates cellular protein and transcriptome measurements at a single-cell level. This method is particularly useful in fields such as cancer, neurobiology, stem cell biology, immunology, and developmental biology, where variations in RNA transcripts and protein expression between cells can be essential to answering important questions.

What is CITE-Seq?

CITE-Seq, or Cellular Indexing of Transcriptomes and Epitopes by Sequencing, is a technique that enables the simultaneous sequencing of RNA and the quantification and characterization of surface proteins using specific antibodies at the level of individual cells. It combines highly multiplexed antibody-based detection of protein markers with unbiased transcriptome profiling for thousands of single cells in parallel.

Principles of CITE-Seq

Cellular Indexing of Transcriptomes and Epitopes (CITE-seq) uses unique oligo-tagged antibodies to identify surface proteins, using sequencing as a readout. The number of barcodes that can be conjugated to antibodies surpasses the number of fluorophores or heavy metal tags used in flow cytometry or CyTOF, expanding the number of proteins that can be measured simultaneously with RNA. These barcoded antibodies are part of a unique workflow that produces protein and nucleic acid data using next-generation sequencing (NGS) technologies.

This:

• helps in the creation of oligo dT-based RNA sequencing libraries
• enables barcode-based antibody identification
• allows specific PCR amplification

The number of reads corresponding to each conjugated DNA barcode reflects the identity and abundance of proteins.

Downstream bioinformatics analysis then provides multimodal information on the state of the cells. Some tools available for CITE-seq single-cell analysis are the CITE-seq Python package and Cellranger. Seurat has also made available a multimodal data analysis pipeline for this application.

Advantages of CITE-Seq

CITE-seq offers several advantages over traditional single-cell RNA sequencing, including the ability to identify cell types and states based on both transcriptomic and proteomic profiles.

1. The cellular contents are barcoded in single cells, which allows scientists to obtain multiple readouts. Therefore, the characterization of cellular phenotypes in detail is easier than by performing transcriptome sequencing alone.
2. Previously, protein detection by flow or mass cytometry required optimized panels of antibodies. CITE-Seq provides no upper limit to the number of antibodies that can be used to identify cell surface proteins.
3. Parallelly quantifying the transcriptome and cell surface proteins of thousand cells reduces the overall cost and time of high-throughput sequencing.
4. CITE-Seq can be adapted to detect small molecules, RNA interference, CRISPR, and other gene editing techniques.

Limitations of CITE-seq

Despite its advantages, CITE-seq also has some limitations:

1. Due to the way the cells are treated, there is a loss of location information.
2. Shared challenges of scRNA-Seq, such as the high noise and possible challenges in detecting lowly expressed genes.
3. In terms of phenotyping, optimization of the assay and antibodies also presents a potential problem if proteins of interest are not included in the currently available panels.
4. CITE-Seq cannot detect intracellular proteins. With currently available protocols, the challenges during the permeabilization step limit the technique to surface markers.

CITE-Seq's ability to simultaneously quantify protein and transcriptomic data within single cells has led to important discoveries in various fields. Researchers from the University of Minnesota have used CITE-Seq to identify that a heterogeneous population of macrophages can prevent heart damage. In breast cancer, CITE-Seq has allowed for the categorization of cells based on their cellular makeup and response to treatment, providing a more comprehensive understanding of the disease. Additionally, CITE-Seq has been used to improve our understanding of mild to moderate COVID-19 disease through the analysis of immune cells.