Single-Cell Sequencing: Best Practices and Advice for Sample Prep

Single-cell sequencing yields valuable gene expression information unobtainable through bulk sequencing. Such information can identify distinct cell types, or reveal the complexity of cellular heterogeneity in tumor microenvironments. While single-cell sequencing has become more common, it remains a complex technique. Single-cell sequencing data quality is highly dependent upon the quality of the starting sample of nucleic acids, so proper sample preparation is paramount. From the first step of cell isolation, through the final step of library preparation prior to next-generation sequencing, many points in the workflow warrant extra attention as places to optimize for high-quality results. This article discusses best practices for sample preparation prior to single-cell sequencing (either DNA or RNA), and offers expert advice for dealing with common challenges.

Cell isolation

When isolating cells for single-cell sequencing, the goal is to obtain enough single, healthy cells for the experiment while minimizing artifacts. Careful handling of tissues is especially important at this stage. “Cell handling can have a significant impact on single-cell RNA assay quality and requires cell dissociation, as cell clumps can manifest as high duplicate rates,” says Robert Meltzer, Senior Director of Advanced Projects at Fluent BioSciences. “To minimize damage to cells, pipette with wide bore pipettes when possible to reduce shear.”

Likewise, pay careful attention to the effects of centrifugation on cell health. “Use swinging bucket rotors when pelleting cells,” says Meltzer. “Fixed angle rotors will cause cells to impact the walls of the sample tube and slide toward the tip of the tube, which can damage cells and decrease sample viability.”

Common isolation methods include fluorescence-activated cell sorting (FACS), which has the highest throughput, and magnetic-activated cell sorting (MACS) for low-to-medium throughput. A less common method is laser capture microdissection (LCM), which is low throughput but enables spatial accuracy when this is necessary to isolate specific cell types from tissues based on anatomical context.

FACS is widely used for isolating and sorting large numbers of single cells prior to sequencing, but it requires attention to cell health. “It’s important to optimize sorting conditions [for FACS] such as speed and run time to ensure sample quality throughout the sorting process,” says Zuleyma Peralta, Product Manager for Sample Preparation at 10x Genomics. “FACS remains a powerful tool for cell isolation, enabling the enrichment of rare cell types and the removal of sample impurities, thereby preparing the cells for successful single-cell sequencing.”

Optimizing FACS parameters such as speed, pressure, and run time can help to preserve cell health during the isolation and sorting process. “Typical FACS-based single-cell sorting utilizes high pressure, which can cause cellular stress or damage, and may therefore result in artifacts in gene expression profile or heightened noise from damaged nucleic acids,” says Tracy Liu, Senior Product Manager at Bio-Techne. “The most common method people use to sort single cells today is FACS, and they are looking for gentler single-cell isolation technologies, like Bio-Techne’s Namocell microfluidics-based sorting instrument.”

MACS is a simple, scalable, affordable alternative to FACS that requires no special instruments, and can process high numbers of cells (albeit less than FACS). “Cell suspensions obtained through MACS may not reach the same purity level as those achieved with FACS, because MACS primarily relies on antibody conjugation for selection without incorporating additional refining conditions,” says Peralta. “However, for some projects, this purity level may be acceptable, and any limitations can be addressed through other cleanup strategies if required.”

10x Genomics offers a wide range of sample-preparation protocols, which can be used as a starting point and optimized for your samples. “For instance, enzymatic incubation times and temperatures can be fine-tuned to optimize tissue dissociation, and lysis concentration and times can be adjusted for nuclei isolation,” says Peralta. “10x Genomics also provides prepackaged sample-preparation solutions, such as the Chromium Nuclei Isolation Kit, which provide optimized workflows across a variety of tissue types and downstream single-cell assays.”

Workflow and library prep kits

After cell isolation, sample enrichment can improve your pool of single cells by removing dead cells or unwanted cell types. “Dead cells can release RNAses [that can degrade your target], and RNA that results in high background and can adversely affect the ability to identify distinct cell types in scRNAseq data,” says Meltzer. “Rather than focusing on improving sample quality after the fact, researchers are advised to improve sample handling for increased cell viability from the outset.”

Library prep kits designed for single-cell sequencing help to streamline the workflow for greater ease-of-use and reduced errors. Suitability for your application is important when choosing a library prep kit. Of paramount concern during the sample-preparation workflow is minimizing both sample loss and potential bias, as both factors can lead to lower data quality. “[Minimizing loss and bias] would require careful evaluation and selection of methods and kits for these procedures,” says Liu. “For instance, whole genome (WGA) or whole transcription amplification (WTA) is often necessary in single-cell sequencing, and the commercially available WGA or WTA kits are not equal.”

Overall, look for kits that maximize library complexity as well as yield. Ideally, library complexity is maintained with less bias and more representation of the genetic material. In addition, a greater yield is focused on minimizing loss and nucleic acid degradation. “Higher yield at each intermediate step helps the library complexity, so it is desirable to have a protocol with fewer pipetting steps and less tube-switching,” says Liu.

Navigating complex samples

When sequencing from single cells, sample types range in complexity from simpler cell suspensions to more challenging tissues. For example, FFPE tissues are difficult to work with for many reasons. Starting materials can be limited, especially with rare or archival samples. In addition, samples might have experienced variations in time prior to fixation, as well as fixation protocols and storage conditions. All of these factors can affect the quality of nucleic acids. “Probe-based approaches, like that used by Chromium Single Cell Gene Expression Flex, offer the advantage of capturing degraded transcripts even in cases where a poly-A tail may be absent,” says Peralta. “This is particularly beneficial as it enables the analysis of archival samples using new technologies, expanding the possibilities for studying historical or preserved samples.”

Organoids, another complex sample type, are composed of certain cell types grown together with the aim of mimicking the structure and physiology of specific tissues in vivo, and are increasingly used as tissue and organ models. “Organoid [samples] can be particularly difficult to prepare due to their small sample size and distinct cell characteristics,” says Peralta. “To overcome these challenges, it is crucial to carefully optimize dissociation protocols and handling conditions to minimize cell stress and maintain cell viability.”

Indeed, the ability to sequence single cells from organoids may prove a useful tool for staging the maturity and functionality of organoids going forward. Increasingly, scientists are using organoids to study models of human diseases. Single-cell sequencing tools may help them further the application of brain organoids, for example, into models of neurodegenerative conditions such as Alzeimers disease.

Key Takeaways

Cell isolation

• Handle tissues carefully to minimize artifacts
• Use wide-bore pipettes during cell dissociation to reduce shear and maintain cell integrity
• Opt for swinging bucket rotors instead of fixed angle rotors during centrifugation to prevent cell damage

Workflow and library prep kits

• Use sample enrichment to remove dead cells and unwanted cell types before library preparation
• Choose library prep kits designed for single-cell sequencing to streamline the workflow and reduce errors
• Look for kits that maximize library complexity, minimize sample loss and bias, and provide a greater yield

Navigating complex samples

• Handle FFPE tissues with probe-based approaches that capture degraded transcripts
• Optimize dissociation protocols and handling conditions for challenging sample types like organoids to maintain cell viability
• Consider single-cell sequencing to assess the maturity and functionality of organoids, particularly in disease modeling