Single-cell RNA sequencing: how it works and applications

A primer on single-cell RNA sequencing

Single-cell RNA sequencing (scRNA-seq) is a cutting-edge technique that allows researchers to study gene expression at the level of individual cells. scRNA-seq has become an essential tool in molecular biology, providing researchers with unprecedented insight into the complexity of biological systems.

Here is a brief description of how scRNA-seq works and some applications.

How does scRNA-seq work?

scRNA-seq involves four primary steps: cell isolation, RNA capture, reverse transcription, and sequencing.

1) Cell isolation

The first step in scRNA-seq is to isolate individual cells from a tissue or culture. Depending on the experimental design, cells can be isolated using various techniques, including fluorescence-activated cell sorting (FACS), microdissection, or micromanipulation.

2) RNA capture

Once individual cells have been isolated, the next step is to capture their RNA. There are two primary methods for RNA capture in scRNA-seq: microfluidic devices and droplet-based methods.

In microfluidic devices, individual cells are captured in microchambers, and RNA is captured using oligo-dT primers immobilized on a solid surface. This method allows for high throughput and capture efficiency but can be expensive and technically challenging.

In droplet-based methods, individual cells are encapsulated in droplets along with oligo-dT beads, lysis buffer, and reverse transcriptase. This method is more cost-effective and more accessible to perform than microfluidics but has lower capture efficiency and a higher rate of doublets (multiple cells in the same droplet).

3) Reverse transcription

After RNA capture, the next step is to convert the RNA into cDNA using reverse transcription. This step involves the addition of reverse transcriptase, which uses oligo-dT primers to synthesize cDNA from the captured RNA.

4) Sequencing

Finally, the cDNA is sequenced using high-throughput sequencing technologies such as Illumina or PacBio. The resulting sequencing data can be analyzed using bioinformatic tools to identify and quantify gene expression in individual cells.

Applications of scRNA-seq

scRNA-seq has numerous applications in various research fields, including developmental biology, cancer research, neuroscience, and immunology. Some of the critical applications of scRNA-seq are described below.

1) Identifying rare cell types

scRNA-seq allows researchers to identify rare cell types that may be overlooked using traditional techniques. For example, scRNA-seq has been used to identify rare cell types in the brain, such as astrocytes and oligodendrocytes, previously challenging to isolate and study.

2) Characterizing cell diversity

scRNA-seq can be used to characterize cellular diversity within a tissue or population of cells. This information can be used to create cell atlases, which provide a comprehensive map of the cellular composition of a tissue or organism.

3) Studying gene regulation

scRNA-seq can be used to study gene regulation at the level of individual cells. By examining the expression of transcription factors and other regulatory genes in individual cells, researchers can gain insights into transcriptional networks underlying cellular function and differentiation.


scRNA-seq has revolutionized the field of genomics and provided insights into the complexity of biological systems. The increased resolution, identification of cellular heterogeneity, and the ability to identify novel cell types and states have all contributed to the advances made in this field. With the development of new computational tools and integration with other omics technologies, the future applications of scRNA-seq are promising.

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