Introduction to Single Cell Sequencing – Cite-Seq – Series 3

May 5, 2023

By Derek Vargas and Dr. Anantha Kethireddy , Scientific Affairs, MedGenome Inc

Cite-Seq, short for Cellular Indexing of Transcriptomes and Epitopes by sequencing, is a powerful technology that has revolutionized single-cell sequencing. With its ability to analyze transcriptomes and protein expression at a single-cell level, Cite-Seq has the potential to greatly advance our understanding of cellular heterogeneity and function in biological systems. In this article, we will discuss the workings of Cite-Seq, its current and potential applications in various fields of research, and its limitations.

How Cite-Seq Works?

Cite-Seq is a technique that combines single-cell RNA sequencing (scRNA-seq) with antibody-based surface protein detection. The goal is to analyze the transcriptome of each individual cell, along with the surface proteins that are expressed on the cell membrane. By doing so, researchers can get a better understanding of the diversity and functionality of individual cells in a population. The Cite-Seq workflow involves several key steps:

  • 1. Surface Protein Staining with Antibody-oligonucleotide Conjugates:
    The first step is to dissociate the tissue into a single cell suspension. Next, the cells are stained with a panel of antibodies targeting specific surface proteins of interest. Each antibody is conjugated to a unique oligonucleotide barcode, which allows for the identification of the protein that is bound to each cell. The cells are then sorted based on the presence or absence of each surface protein, and the RNA and protein are isolated from each individual cell.
  • 2. Single Cell RNA Sequencing & Cell surface protein detection:
    The next step is to generate gel bead in emulsion (GEM) with antibody labeled cells. Because the antibodies attached to the individual cell, they end up together in one GEM. This can be done using a droplet-based method developed by 10X Genomics. The cells are encapsulated in tiny droplets, along with a bead that contains a unique barcode. The reverse transcriptase then adds the barcode into the mRNA transcripts of the cell, allowing for its identification during downstream analysis. The cell barcodes are also added to the antibody-oligonucleotide conjugate.
  • 3. Sequencing and Data Analysis:
    The RNA and protein from each individual cell are then sequenced using standard techniques. The sequencing data is then analyzed using bioinformatic tools that allow for the identification of individual cells based on their gene expression and protein markers. By analyzing the transcriptomes and protein expression of individual cells, researchers can identify new cell types, characterize the heterogeneity of cell populations, and study the relationships between different cell types.
  • 4. Advantages of CITE-SEQ:
    Link a cell’s RNA profile with its surface proteins. Profiles multiple surface proteins simultaneously. Combines long standing knowledge of surface protein analysis with ever more complete RNA -Seq data.

Applications of Cite-Seq in Biological Research

Cite-Seq has a wide range of applications in biological research. One of the most significant applications is in the identification of new cell types and the characterization of cellular heterogeneity. By analyzing the transcriptomes and protein expression of individual cells, researchers can identify rare or previously unknown cell types and explore the differences between cell populations. This has important implications for understanding disease states and developing new treatments.

For example, Cite-Seq has been used to identify new immune cell subsets and characterize their roles in the immune response. Researchers used Cite-Seq to investigate the heterogeneity of T cells in the lung tissue of mice infected with influenza virus. They identified a new subset of T cells that expressed a specific set of surface proteins and had a unique gene expression profile. This subset was found to be important for controlling viral replication and preventing lung tissue damage.

Cite-Seq has also been used to investigate the differentiation of stem cells into specific cell types. By analyzing the transcriptomes and protein expression of individual cells during the differentiation process, researchers can identify the genes and proteins that are important for cell fate determination. This has important implications for regenerative medicine and the development of cell-based therapies.

MedGenome offers end-to-end project support for Cite-Seq (TotalSeq A, B & C ) experiments. Our high-throughput lab can take fresh tissue samples, dissociate them into single-cell suspensions, then stain with oligonucleotide-conjugated antibodies (we recommend Biolegend’s universal cocktail for maximum coverage). We generate scRNA-Seq libraries using the 10X Genomics platform.

Our bioinformatics team is specialized in providing cutting-edge analysis of single-cell data, using the latest technology and techniques to help you gain deep insights into your biological samples. Whether you are working in genomics, transcriptomics, or other fields, our team of expert analysts can help you interpret your data with precision and efficiency. We use advanced algorithms and machine learning techniques to analyze your data and provide customized reports that meet your unique needs. With our comprehensive approach to single-cell data analysis, you can be sure that you are getting the most accurate and reliable results possible.


#Cite-Seq, #for Cite-Seq experiments, #scRNA-Seq libraries, #Surface Protein Staining, #Antibody-oligonucleotide Conjugates, #single-cell data analysis


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