
New methods seem to emerge almost weekly in the rapidly evolving field of single-cell sequencing. This abundance of options can be daunting for researchers as they try to find the best technique for their specific needs. Fortunately, Contract Research Organizations (CROs) like Single Cell Discoveries offer the expertise and experience to help navigate this complex landscape. In this blog, we will look at two main types of single-cell sequencing methods: instrument-free and instrument-based.
What is Single-Cell RNA sequencing?
Single-cell RNA sequencing (scRNA-seq) identifies the RNA content from individual cells using optimized next-generation sequencing technologies. This approach provides a higher resolution of cellular differences and a better understanding of the function of individual cells within their microenvironment. In a nutshell, it involves several key steps:
- Preparation of single cell suspension.
- Extraction, processing, and amplification of the RNA from each isolated cell.
- Preparation of a sequencing library.
- Sequencing of the library using a next-generation sequencer.
Instrument-Free Sequencing Methods
The shift towards instrument-free single-cell sequencing is evident with recent additions to the landscape, detailed in this section.
Scale Biosciences
Scale Biosciences (Scale Bio) uses a split-pool combinatorial barcoding technique for single-cell RNA sequencing. This method involves multiple rounds of splitting and pooling cells, where each round randomly adds a new barcode to the RNA molecules within the cells. The combination of randomly assigned barcodes creates a unique code for each single cell. This process allows for high-throughput 3’ single-cell RNA sequencing without the need for complex instrumentation. The key advantages include scalability, cost-effectiveness, and the ability to process large numbers of cells simultaneously.
Scale Biosciences. "Streamlined Workflow." Scale Biosciences. Accessed January 31, 2025. https://scale.bio/single-cell-rna-sequencing-kit/
Click here to learn how Single Cell Discoveries leverages high throughput combinatorial single-cell sequencing as a Scale Biosciences Service & Technology Endorsed Provider.
Watch our webinar with Scale Bio: CRISPR screening at scale
Achieving 1 million cell data sets with an instrument-free, cost-effective approach.
Parse Biosciences
Parse Biosciences has introduced Evercode™ technology, which also relies on split-pool combinatorial barcoding. It allows researchers to label millions of cells or nuclei with unique barcodes. Differently from Scale Bioscience, Parse additionally uses random priming to increase gene body coverage.
The process involves fixing and permeabilizing cells, followed by multiple rounds of barcoding within the cells themselves. This technique offers high sensitivity, scalability, and flexibility, making it suitable for large-scale single-cell projects. Single Cell Discoveries offers a range of services utilizing the advanced technologies developed by Parse Biosciences. Click here to talk to our experts about how we can utilize combinatorial barcoding to help with your research question.
Fluent BioSciences/Illumina
Fluent BioSciences and Illumina utilize a chip-free droplet emulsion method for single-cell RNA sequencing. This technique involves creating droplets containing single cells, lysis buffer, and barcoded primers without the need for microfluidic chips. The droplets act as individual reaction vessels, allowing for the capture and sequencing of RNA from single cells. This method simplifies the workflow and reduces costs while maintaining high data quality.
Clark, Ian C., et al. "Microfluidics-Free Single-Cell Genomics with Templated Emulsification." Nature Biotechnology, 2023, https://doi.org/10.1038/s41587-023-01685-z. Figure 1a-d.
Universal Sequencing Technology
AmpliDrop by Universal Sequencing Technology also uses a chip-free droplet emulsion approach. This method involves fixing cells using formaldehyde before encapsulating single cells in droplets, barcoded primers, and reagents for RNA capture and sequencing.
The process is designed to be user-friendly and scalable. It has low setup costs and efficient cell sequencing. AmpliDrop is particularly useful for identifying rare cell populations and comparing cell states within heterogeneous samples.
CSgenetics
CSgenetics offers the SimpleCell™ platform, which uses a viscous matrix or 'liquid suspension' method for single-cell RNA sequencing. This technique involves capturing cells paired with a cell-binding bead containing barcoded primers in a viscous liquid. This allows for capturing and sequencing RNA easily. This method is cost-effective and suitable for a wide range of sample types, including blood, cell lines, and disaggregated tissues. Most noticeably, changing the specificity of the cell binding beads allows for selective sequencing of cell types of interest.
CS Genetics. "Workflow Image." CS Genetics. Accessed January 31, 2025. https://www.csgenetics.com/product/
BioSkryb
BioSkryb’s ResolveOME™ uses Primary Template-Directed Amplification (PTA) for high-fidelity whole genome and transcriptome amplification without relying on complex instruments. PTA is an isothermal method that selectively amplifies DNA from single cells or low-input samples at a single temperature, avoiding the need for thermocyclers or microfluidic systems. This is combined with full-transcript reverse transcription, and BaseJumper® computational tools to generate comprehensive single-cell multiomic analysis.
BioSkryb offers pre-optimized kits that include all the needed reagents. This helps researchers get accurate and uniform genome coverage, as they can do this using simple lab tools like pipettes and incubators.
BioSkryb Genomics. "Figure 1. The Principle of PTA." In "ResolveDNA® Integration with Illumina DNA Prep and DNA Prep with Enrichment to Enable Single-cell Genomics." BioSkryb Genomics. Accessed January 31, 2025. https://www.bioskryb.com/resolvedna-integration-with-illumina-dna-prep-and-dna-prep-with-enrichment-to-enable-single-cell-genomics/
Efficient and cost-effective
All things considered, instrument-free sequencing is a revolutionary advancement in single-cell RNA sequencing, enabling high-throughput analyses without the need for complex instrumentation. This method uses techniques like split-pool combinatorial barcoding and chip-free droplet emulsions. It allows for efficient and cost-effective processing of large groups of cells.
The main benefit is that it removes the need for special hardware. It also lowers the risk of failure. Additionally, it makes distributing test kits easier, which makes the technology more accessible.
Instrument-Based Sequencing Methods
Instrument-based methods in sequencing use advanced tools like microfluidic devices and automated platforms. These tools help to make analyses more precise and efficient. These systems excel in generating high-quality data with minimal variability, thanks to their controlled environments and optimized workflows.
The main advantage of instrument-based sequencing is that it allows for faster turnaround times, as automated workflows minimize manual handling and streamline data acquisition. While instrument-free methods are more flexible and potentially cheaper, they can become less cost-effective at very high throughput levels due to increased labor.
10x Genomics (Chromium)
The 10x Genomics Chromium platform is a microfluidics-based system widely used for single-cell RNA sequencing. The platform partitions single cells into oil droplets, each containing barcoded gel beads with oligonucleotides. These barcodes uniquely label the RNA transcripts from individual cells, enabling high-resolution data analysis.
Chromium supports a wide range of applications, including single-cell gene expression, immune profiling, and multiomic assays that integrate gene expression with chromatin accessibility or protein expression. Its scalability, ease of use, and strong data quality have made it essential in single-cell research. New versions and protocols keep expanding its capabilities.
We offer 10x Genomics Single Cell Gene Expression, Single Cell Immune Profiling, and Single Cell Gene Expression Flex as a service. Click here to find out how we effortlessly analyze thousands of cells in high throughput as a 10x Genomics Certified Service Provider.
Get the 10x Genomics
information guide
You can find a full explanation of 10x Genomics, its variations, and our approach in our information guide.
BD Rhapsody
BD Rhapsody is an instrument and nanowell-based platform for single-cell RNA sequencing. The process involves isolating cells and beads in microwells, where RNA is captured using barcoded polyT primers.
This versatile platform supports a wide array of assays, making it adaptable to diverse research needs. The multiome option combines different -omics layers. This is a unique feature that may allow for higher throughput in future versions.
Which sequencing method serves your biological question?
When deciding between instrument-free and instrument-based methods for single-cell analysis, researchers must consider the specific biological questions they aim to address. Instrument-free methods, such as split pool-based systems, offer flexibility and can be more cost-effective, making them appealing to labs with limited resources. These methods allow for rapid experimentation.
On the other hand, instrument-based methods like BD Rhapsody provide higher throughput, enabling a multisample workflow. The ability to integrate multiple -omics layers in a single experiment can reveal complex interactions within biological systems that might be missed with simpler approaches.
Ultimately, the choice of method depends on a few factors. These include the research goals, the resources available, and the level of detail needed in the data. For instance, if the goal is to explore gene expression patterns across a large population of cells, an instrument-free approach could be more cost-effective. Conversely, if the focus is on preliminary screening or exploring many different modalities studies, instrument-based methods would be better.
Both methods have their benefits. The knowledge of CROs like Single Cell Discoveries can help you choose the best option for your biological questions.
Click here to book a meeting to explore which sequencing service is most suitable for your research.