CITE-seq, or Cellular Indexing of Transcriptomes and Epitopes by sequencing, allows scientists to analyze individual cells in detail. This technology simultaneously measures two types of molecules from a single cell: its RNA messages and its surface proteins. By combining these two layers of biological information, CITE-seq offers a more complete understanding of a cell’s identity and its roles within complex biological systems. This integrated view helps researchers identify specific cell types and understand their functional states more accurately.
The Foundation of Single-Cell Analysis
Before CITE-seq, single-cell RNA sequencing (scRNA-seq) advanced our ability to study individual cells. ScRNA-seq focuses on the transcriptome, which includes all RNA molecules in a cell. These RNA molecules are the cell’s internal instruction manuals, dictating which proteins should be made. By analyzing the transcriptome of thousands of individual cells, researchers can identify different cell types within a tissue and understand their gene expression patterns.
Imagine trying to understand how a factory operates by only reading the instruction manuals provided to each worker. ScRNA-seq provides this level of detail, showing what instructions each individual cell has received. Despite its power, scRNA-seq primarily reveals the instructions (RNA) rather than the actual tools being used (proteins). Proteins perform most of a cell’s functions, acting as enzymes, structural components, or signaling molecules on the cell surface. Relying solely on RNA data might not fully capture a cell’s true functional state or identity.
How CITE-seq Works
CITE-seq addresses the limitation of traditional scRNA-seq by measuring cell surface proteins. It uses specially designed antibodies that bind to specific target molecules. Each antibody is tagged with a unique DNA barcode.
In the CITE-seq process, scientists introduce these DNA-barcoded antibodies to a cell sample. The antibodies attach to their corresponding proteins on each cell’s surface, labeling them. After labeling, cells are prepared for sequencing, similar to a standard scRNA-seq workflow. During sequencing, both the cell’s RNA and the DNA barcodes from bound antibodies are read. This results in two distinct datasets: one detailing RNA expression and another quantifying surface protein abundance.
Integrating Protein and Gene Expression Data
The strength of CITE-seq comes from simultaneously measuring and integrating both RNA and protein data from each cell. This combined dataset provides a more precise understanding of a cell’s state. RNA levels do not always directly correlate with protein levels, as cellular mechanisms can regulate protein production and degradation after RNA is transcribed. For instance, a cell might have high levels of RNA for a particular protein but produce very little of the actual protein due to post-transcriptional control.
By measuring both RNA and protein, CITE-seq resolves this ambiguity, offering a clearer picture of a cell’s functional identity. This dual profiling allows researchers to identify cell types that might appear similar based on their RNA profiles alone but are functionally distinct due to differences in their surface protein expression. This integrated view enables more accurate cell classification and characterization.
Real-World Research Applications
CITE-seq is a valuable tool across various fields, providing insights into complex biological systems. In immunology, CITE-seq creates maps of immune cell populations. Researchers identify rare immune cell subtypes and understand how immune cells respond to infections, vaccines, or autoimmune diseases by simultaneously profiling their gene expression and surface markers. This helps dissect cellular interactions that support immune responses.
In cancer research, CITE-seq helps scientists dissect the heterogeneous environment of tumors. Tumors contain a complex mix of immune cells, stromal cells, and blood vessels that interact to influence disease progression and treatment response. CITE-seq allows for detailed characterization of these diverse cell populations within the tumor microenvironment, revealing how cancer cells evade the immune system or how immunotherapies affect cellular interactions. This detail assists in developing more targeted and effective cancer treatments.
CITE-seq also contributes to developmental biology by tracking cellular differentiation processes. As stem cells mature into specialized cell types, their gene expression patterns and surface protein profiles change dynamically. CITE-seq provides a dual-layer view of these transitions, capturing both the evolving genetic programs (RNA) and the emergence of specific cell identities (surface proteins). This helps researchers understand the molecular events that govern cell fate decisions and tissue formation during development.