Researchers planning to employ single-cell transcriptome sequencing face a few complex choices.
First, dozens of technologies enable single-cell transcriptome sequencing, each with different ups and downsides. How do you make an informed selection, and how do you progress toward a final decision?
Then, depending on the technology, there is a practical step to consider. You can do the experiments yourself, employ a core facility, or outsource to a service provider. Which option would bring your project the best results?
Finally, you have myriad options to consider for the experimental setup, which are often a trade-off between quality and costs. Larger and smaller tweaks come with proportionate effects, but there is always one best solution to suit your needs. What should you consider in your experimental setup plan?
This blog guides you through some of the most multifactorial choices in your research. Click below to jump to a specific section:
- Which single-cell transcriptome sequencing technology to use?
- Why consider outsourcing single-cell transcriptome sequencing?
- Estimate the costs of your single-cell sequencing experiment
- Our advice
For a refresher on single-cell transcriptome sequencing, read our blog post “What is Single Cell Transcriptomics?” It covers the basics, mechanisms, history, and applications of single-cell transcriptomics.
Which single cell transcriptome sequencing technology to use?
There are various technologies for single-cell sequencing projects. The right choice depends on your specific biological question. Each has its benefits. This is how they compare.
It is important to know that, together, the technologies discussed below can accomplish almost everything single-cell sequencing has to offer. If one technology seems unable to answer your needs, one of the other technologies may have all the requirements.
Need advice on the best technology for your project? Select the most suitable technology in our service selection tool.
Robotic lab automation speed up plate-based services (SORT-seq and VASA-seq)
SORT-seq is a sensitive and versatile single-cell sequencing technology based on FACS sorting and 384-well cell-capture plates. Start by sorting one live cell into each well of the capture plate, which has specific primers with barcodes. After sorting, you freeze the plates and send them to us for library prep and sequencing.
Key advantages of SORT-seq:
- Low input: SORT-seq is a single-cell sequencing method that is ideal for small populations. It uses only 380 cells per plate, which makes it a low-input method.
- Flexible: Because of the modular nature of working with plates, you can start small and scale your experiment later.
- Specificity: FACS sorting gives total control over the cell type you sort into the plates. This enables both basic, unbiased sorts, and complex sorts of specific populations of interest.
- Handle many cell types: SORT-seq can analyze larger cell types, such as cardiomyocytes, without issues. This is similar to 10x Genomics Gene Expression Flex, yet is not limited to mouse and human cell types.
- Perfect for small sample sizes: all single-cell sequencing methods lose cells between sampling and data analysis. SORT-seq can handle small amounts of starting material, such as a few thousand cells from your starting tissue. This is helpful when you do not have millions of cells to spare.
VASA-seq is another technology based on FACS sorting and 384-well plates. It’s an exclusive technology that does not rely on the presence of a poly-A tail in transcripts. This allows for full-length and total RNA single-cell sequencing, including non-coding and premature RNAs.
VASA-seq provides full-length sequencing information on both coding and non-coding RNA species.
The process is similar to SORT-seq: sort single cells into the wells of our 384-well cell capture plates, send them to us, and we perform the single-cell transcriptome analysis.
Key advantages of VASA-seq:
- More RNA species: Full-length sequencing enables analysis of sequence variation and splice alternatives.
- VASA-seq can analyze non-coding RNA species such as snoRNAs, snRNA, and scaRNAs.
- Sensitivity: VASA-seq is one of the most sensitive single-cell sequencing technologies.
The 10x Chromium X microfluidics device.
10x Genomics designs and manufactures devices and reagents for single-cell transcriptome analysis. Their Chromium Controller (Chromium X is the newest version) is a microfluidics device that enables high-throughput single-cell sequencing for thousands of cells at a time.
We offer three 10x Genomics single-cell transcriptomics products at Single Cell Discoveries. These products are Single Cell Gene Expression, Single Cell Gene Expression Flex, and Single Cell Immune Profiling.
Single Cell Gene Expression
With the Single Cell Gene Expression solution, we generate 3’ single-cell transcriptomics data for up to 10,000 cells per lane or sample. This provides a high-throughput solution for experiments of any scale.
Single Cell Gene Expression Flex
Single Cell Gene Expression Flex is an entirely new protocol that enables single-cell sequencing of fixed tissues. It answers to a widespread need in preclinical and clinical research to maintain tissue quality during transportation and storage. SORT- and VASA-seq can work with acetic-methanol-fixed cells, and other 10x Genomics technologies work with methanol-fixed cells. Yet for other popular methods for fixation, like formaldehyde or FFPE, analysis requires Single Cell Gene Expression Flex.
The technology utilizes specifically designed RNA probes to capture enough of the transcriptome of single cells to do the analysis. This affects accuracy minimally. However, the probes are specifically for humans and mice, so sequencing formaldehyde-fixed tissues of other species is impossible.
Single Cell Immune Profiling
This solution generates single-cell transcriptomics data of mRNAs from the 5′ end. The data thus includes the V(D)J sequences in antibodies and T cell receptors. Scientists often use this technique to characterize the immune repertoire from the T-cell or B-cell receptors. This means you can measure the diversity in your immune cells and their response to pathogens or drugs with great accuracy.
Key advantages of 10x Genomics:
- High-throughput: The 10x Genomics platform enables the analysis of thousands of cells per sample. Large experiments like in preclinical research are very suitable for 10x Genomics.
- Leading technology: The 10x Genomics single-cell platform appears in over 6000 peer-reviewed publications.
- Standardized: The commercially available 10x Genomics system is highly standardized, ensuring consistent results.
- Flex: work with formaldehyde-fixed tissue.
For more information about these technologies, visit their respective pages by clicking the links or find an overview in our information guide.
10x Genomics solutions can acquire additional information because of a technology trait called feature barcoding. It enables single-cell barcoding of some other elements in a cell, such as the cell-surface proteins. In the field, we often refer to this latter solution as CITE-seq.
Read more about CITE-seq here.
Moreover, some research questions require targeted amplification of a gene or RNA transcript. It is often possible to combine single-cell transcriptome sequencing with targeted amplification.
In addition, single-cell sequencing is compatible with CRISPR screens. Designed CRISPR guides are added to tissues that create many genetic perturbations. In the context of a stimulus such as a drug, the cells will respond differently depending on the perturbations. Researchers can study the stimulus effects and interplay with genes by capturing the single-cell gene expression profiles and the CRISPR guide sequences.
Finally, it is possible to combine a single-cell sequencing experiment with Single Cell ATAC. This technique captures the chromatin accessibility profile of single cells.
Read more about Single Cell ATAC here.
You can find the best technology for your project together with our PhD level experts. Just ask your question in the chat or file a contact form to find your answer.
Bespoke single-cell solutions
Projects that do not fit the requirements of the technologies discussed above are not necessarily impossible. Our R&D unit is eager to work on your non-standard project, whether it is a novel application, a tough tissue, or an uncommon species that requires technological tinkering.
Read more about our Research & Development approach.
Why consider outsourcing single cell transcriptome sequencing?
While settling on a technology to use, there is another choice to make. Setting up a single-cell sequencing department can be challenging because of specialized resource requirements. Acquiring skilled staff, sufficient space, and investing time in training can be difficult. Instead, you can consider outsourcing some of your single-cell sequencing projects.
There are actually three options for single-cell sequencing: use a core facility, outsource your project, or create your own department. Here, we explain these three options.
Large institutes can have a core facility dedicated to single-cell sequencing or dedicated to genomics in general. Many researchers choose their local core facility because of its convenient location and easy access. Additionally, they may already be familiar with the individuals who oversee its operations. Often, they can assist you in the data analysis as well.
However, core facilities may have limited capacity. Additionally, they may not offer all single-cell techniques (see the chapter above) or restrain themselves to relatively standard experiments. Most core facilities offer the most widespread platform, 10x Genomics, but may not have experience with all its extensions.
Meanwhile, scientists in industry often do not have the option to collaborate with an academic core facility. In addition, single-cell sequencing in drug development contexts is often relatively large in scale. Preclinical tests such as safety assessment, drug assays, novel capsid design experiments, et cetera, are generally high throughput. Effective single-cell sequencing at such a scale requires advanced equipment such as liquid handling robots and microfluidics devices.
CRO or service provider
Outsourcing your single-cell sequencing projects allows you to begin immediately without acquiring resources first.
Service providers in the rapidly growing single-cell sequencing field often offer the 10x Genomics platform. Service providers vary in price, speed, scale, and extras. You might prefer one that delivers fast for projects with high time pressure. Yet, not many service providers can deal with large-scale projects or unusual requests.
At Single Cell Discoveries, we specialize in comprehensive single-cell projects, guiding clients from project design to data analysis. This approach is ideal for scientists who want to outsource everything, need more guidance, or work on challenging single-cell projects. Our services include optimizing experiments with pilot runs and providing complete data analysis through our skilled data analysis department.
Furthermore, we are a Certified Service Provider for 10x Genomics, ensuring compliance with their standards when performing your experiment.
In addition, by partnering with us, you can access our exclusive and newly developed technologies.
If you need help with single-cell sequencing, meet our specialists to discuss your choices. Read more about our white-glove approach and client relationships. Or find one of our case studies detailing how outsourcing their single-cell experiments worked for them.
Do it all yourself
Some scientists opt for in-house single-cell sequencing. There are various reasons to make this choice, such as complete control over samples and projects. If you’re planning extensive ongoing single-cell work, purchasing a 10x Genomics Chromium Controller, for instance, could be more suitable. As mentioned, auxiliary automation equipment may also be advisable, and data analysis expertise is another important requisite.
Make your choice
Your choice depends on your preferences and project requirements among these three options for single-cell sequencing.
In our general information guide, we provide an in-depth overview of our approach and services.
Estimate costs of single cell transcriptome sequencing
Between one single-cell experiment and another, there can be huge differences in setup, protocol steps, and costs. Moreover, careful experiment design can help optimize costs. Learn which factors affect the cost of your single-cell project and estimate the value of these factors to your research.
Single-cell sequencing requires separate reactions for each cell, resulting in the need for more reagents compared to bulk experiments. Generally, reagent costs for standard single-cell RNA sequencing experiments are 10 to 20 times higher than comparable bulk RNA experiments.
Next generation sequencing
Single-cell sequencing requires more sequencing reads than bulk RNA sequencing. A typical bulk RNA sequencing analysis needs up to 20 million sequencing reads per sample. Single-cell sequencing requires 50,000 to 150,000 reads per cell. Thus, sequencing costs for single-cell sequencing are approximately 7 to 15 times higher than traditional bulk analyses.
High-throughput next generation sequencing on the NovaSeq X Plus
Regardless of the platform or technology, single-cell sequencing is more sophisticated than conventional molecular biology technologies like bulk RNA sequencing. This inherently raises costs.
Moreover, multiplex assays can bring additional levels of data to your experiment, which may be crucial in answering your biological question.
In addition, the costs depend on the number of samples, target cells per sample, and reads per cell.
Number of samples
The more samples you analyze, the higher the project cost. It is essential to assess your experiment setup critically. Optimize which patients, tissues, or time points to include.
Of course, there are a lot of unknown factors in any experiment. We thus advise starting with a smaller single-cell experiment and scaling up later. Bulk RNA sequencing lends itself well to getting a lower-resolution breakdown of a sample or answering specific research questions. In addition, SORT-seq operates at very low input; it enables single-cell sequencing without using up much precious tissue.
Target cells per sample
Higher cell numbers lead to a more expensive project unless balanced against fewer reads per cell. For instance, with the SORT-seq system, adding more cells necessitates additional plates, increasing costs. With the 10x Genomics system, processing more cells per sample can significantly impact sequencing costs. Consider your project and the biological question to make informed decisions.
Reads per cell
Increased sequencing depth raises experiment costs. Tailor sequencing depth to your specific experiment needs. For cell type identification, shallow sequencing may suffice, while rare gene expression studies may require deeper sequencing. Our experienced staff can guide you in making the best setup decisions for this aspect of your project.
The best advice is to schedule a call to review the variables in your project setup. Our PhD-level experts are keen to help with your setup, whether you have a exact plan or are exploring options.
Ask any questions you have about the services we offer in the chat or with a contact form. Or bring your ideas for non-standard experiments to our team.
Learn everything about single-cell transcriptome sequencing in our information guide:
Get our information guide