Single-cell RNA sequencing has opened the door for many new research questions and pharmaceutical solutions. But what is different from previous RNA sequencing approaches? We want to explain the basics of single-cell RNA sequencing and show its differences from bulk RNA sequencing.
When we think of our body, many cells within a particular tissue type look the same at first glance. Let´s assume you take a sample of this tissue. What conclusions can be drawn from the classical bulk approach? And what from single-cell RNA sequencing? Please have a look at figure 1: It shows that bulk RNA sequencing measures the average expression level of individual genes of the sample’s cells. However, cells in samples often have different functions and, consequently, different expression patterns. That is where single-cell RNA sequencing comes into the picture. It enables the resolution of gene expression on a single-cell level. Thus, differences in cell types can be distinguished within a sample.
Figure 1 | The difference between bulk RNA sequencing and single-cell RNA sequencing.
The differences in single-cell expression patterns cannot be distinguished with bulk RNA sequencing. In contrast, single-cell RNA sequencing enables gene expression resolution on a single-cell level.
But how does it work? An exemplary workflow is shown in figure 2. The single-cell RNA sequencing method uses microfluidics to partition single cells. So-called gel beads contain barcoded oligonucleotides. In the ChromiumTM X instrument, the gel beads are combined with cells and enzymes. This combination occurs in a single aqueous stream within a microfluidic chip channel. Droplets emerge at the end of this aqueous stream as the formed complexes pass the recovery oil interface. These droplets can be seen as reaction vesicles and are called gel beads in emulsion, short GEMs. Those GEMs that contain single cells are collected. The single cells are lysed to enable enzyme reactions within the GEM vesicle, such as reverse transcription or ligation. As the reaction occurs within the GEM, which holds a unique barcode, each cDNA has the same unique barcode. Eventually, the gel beads are dissolved, and the oil is removed to free the barcoded cDNAs into the solution. A pool of cDNAs from multiple cells is formed. As each cell has its own barcode, each cDNA in the pool can later be sorted back to its cell’s origin, allowing analyses on a single-cell level. The subsequent preparation of the NGS library from the barcoded cDNAs can be carried out simultaneously for all nucleotides in the pool.
Figure 2 | Exemplary single-cell RNA sequencing workflow on the ChromiumTM X system.
Both approaches, single-cell RNA sequencing and bulk RNA sequencing, have their strengths and weaknesses, resulting in different application areas for each method. Bulk RNA sequencing approaches are less labor-intensive and time-consuming than single-cell RNA sequencing approaches. The application fields are quite variable and include the detection of alternative splicing and previously unknown transcripts, as well as the identification of novel biomarkers for cancer and other diseases. However, the obtained results cannot be traced back to a single cell type within the sample. The strength of single-cell RNA sequencing makes investigating a cell population’s heterogeneity possible. Further, it enables us to see how cell types interact with each other, how their state changes in different conditions, or how cells transition from one state to another. However, the method is significantly more time-consuming and cost-intensive than bulk RNA sequencing.
Consequently, single-cell RNA sequencing comes into play when investigations require the evaluation of expression patterns at single-cell resolution. Both single-cell RNA sequencing and bulk RNA sequencing approaches offer a wide range of different application fields. Do you want to know which method best suits your research question? We are happy to help you find out!