Single-Cell Sequencing Kits: Choosing the Right Reagents for Your Research
Introduction: Why Single-Cell Resolution Matters
In traditional bulk sequencing, data represents average signals from thousands or millions of cells, potentially masking important differences between individual cells. In contrast, single-cell sequencing uncovers the cellular heterogeneity within a sample – even cells of the same type can vary widely in gene expression and state.. By profiling each cell separately, researchers can identify rare cell subpopulations, map developmental trajectories, and gain insights into complex tissues that would be impossible to detect with bulk methods. This single-cell resolution has proven crucial in fields like cancer biology, immunology, and neuroscience, where understanding individual cell differences can reveal new biomarkers and therapeutic targets
Recent advances have made it feasible to sequence tens of thousands of individual cells in one experiment, providing an unprecedented view of cell-to-cell variation. Single-cell approaches are also expanding into multi-omics, simultaneously measuring genomes, epigenomes, transcriptomes, or proteomes from the same cell to build a more comprehensive picture of biology
Why do reagents matter?
Single-cell sequencing requires specialized consumables at every step, from handling delicate single cells to preparing sequencing libraries from picogram quantities of DNA/RNA. Using the right single-cell sequencing consumables – optimized for low-input and high-throughput performance – is essential to obtaining high-quality data. Below, we outline a typical single-cell RNA sequencing (scRNA-seq) workflow and discuss key challenges at each step, along with the types of reagents and kits that can help overcome those challenges. Whether you are new to single-cell experiments or looking to optimize your protocol, understanding these workflow steps will guide you in choosing the right tools for your research.
Single-Cell Sequencing Workflow Overview
A typical single-cell sequencing experiment involves several sequential steps, each with its own technical considerations. In an scRNA-seq workflow, for example, you would generally proceed through the following stages:
Sample Preparation
Obtain a suspension of viable single cells from your sample (tissue or culture), ensuring cells are healthy and individualized.
Single-Cell Capture
Isolate individual cells into separate partitions (droplets, wells, or microchambers) so each cell can be processed independently.
Barcoding & Reverse Transcription
Lyse the cells and convert their RNA to cDNA, adding unique molecular barcodes so that cDNA from each cell (and each RNA molecule) is tagged with a cell-specific and molecule-specific identifier.
cDNA Amplification
Amplify the minute amounts of cDNA obtained from each cell to yield enough material for library construction.
Library Preparation
Construct sequencing libraries by adding adapters and indices to the amplified cDNA, making the DNA fragments compatible with next-generation sequencing (NGS) platforms.
Sequencing & Analysis
Sequence the pooled libraries on an NGS instrument, then perform bioinformatic analysis to separate reads by cell barcode and interpret the data (quality control, clustering, differential expression, etc.).
Each of these steps must be optimized to retain the fidelity of single-cell information. Below, we delve into each step, highlighting common challenges and the reagent choices that can make or break your single-cell sequencing experiment.
Sample Preparation: Ensuring Viable Single Cells
Everything starts with the quality of your cell suspension. The goal is to isolate individual cells (or nuclei) from tissue or culture while maintaining their viability and RNA integrity. Cell viability is critical – dead or dying cells can release RNA that contaminates your sample, creating “ambient RNA” background noise in droplets. This ambient nucleic acid can be co-encapsulated with healthy cells and lower the signal-to-noise ratio of your data, so you want to minimize cell death before starting single-cell capture. To tackle this, researchers often use cell viability dyes and assays to assess sample quality before loading cells. For example, a simple Trypan Blue exclusion test or fluorescent viability stain can check that a high percentage of cells are alive. If viability is low, you might enrich live cells (and remove debris) using purification kits or gentle centrifugation steps. Additionally, including an RNAse inhibitor in your buffer and keeping cells on ice can prevent RNA degradation during handling.
Another challenge is obtaining truly single cells without clumps or doublets (two cells stuck together). Clumped cells can lead to multiplets being captured in what is supposed to be a single-cell partition. Strategies like gentle trituration (pipetting) and passing the suspension through cell strainers (e.g. 40 μm filters) help create a uniform single-cell suspension. In some cases, enzymatic tissue dissociation kits are needed to disaggregate solid tissues into single cells. Throughout this process, maintaining cells in a buffered solution (with BSA or other carrier protein to prevent cell sticking) can improve recovery.
Key consumables for this stage include cell dissociation reagents (if working from tissue), cell viability dyes (to measure live/dead cell ratios), and sample prep buffers optimized for single-cell genomics. Flow cytometry is also a valuable quality control step – by staining with viability dyes and analyzing forward/side scatter, you can quantify viable cells and even detect doublets or aggregates before sequencing. High viability (typically > 85%) and an absence of clumps or excessive debris set the foundation for a successful single-cell experiment. Avantor offers many of these upstream consumables – for instance, viability/cytotoxicity assay kits and gentle dissociation enzymes – to help you start with the healthiest possible cells.
Single-Cell Capture: Isolating Individual Cells
Once you have a clean single-cell suspension, the next step is to physically isolate each cell into its own reaction environment along with the reagents that will process it. Several technologies exist for single-cell capture, and each has associated consumables:
Microfluidic Droplet Systems
One popular approach uses microfluidic chips to encapsulate single cells into nanoliter oil droplets along with beads and lysis/barcoding reagents. For example, in the droplet method used by 10x Genomics Chromium (a widely used platform), cells are suspended in an aqueous solution and mixed with barcoded gel beads and enzymes in a microfluidic device. Oil is flowed in to compartmentalize this mixture into thousands of droplets, each ideally containing one cell and one bead. The bead carries DNA oligonucleotides with cell-specific barcodes (and often unique molecular identifiers, UMIs), so when the cell is lysed inside that droplet, its RNA molecules all get tagged with a barcode unique to that bead (hence unique to that cell). Droplet generation consumables include the microfluidic chip or cartridge, specialized surfactant oils, and often a specific kit of reagents (buffer, enzyme mix, barcoded bead set) provided by the platform manufacturer. A key challenge here is avoiding doublets, where two cells accidentally end up in the same droplet. To minimize this, protocols dilute the cells to a Poisson distribution – loading at a low concentration so that most droplets have either zero or one cell (Of course, lower loading also means you may need more total cells to achieve a given throughput.) Even with precautions, doublets can occur and have been reported at rates of several percent – in some platforms as high as ~10% or more under high-loading conditions
High-quality microfluidic consumables and careful calibration of cell density help reduce multiplets. For droplet workflows, Avantor provides droplet generation oils and reagents compatible with common platforms, as well as cleaning and calibration supplies for microfluidic instruments.
Microwell Plates and Microchamber Systems
Another capture strategy uses arrays of tiny wells or chambers to physically separate cells. For instance, platforms like BD Rhapsody or Takara ICELL8 use nanowell chips – you pipette a diluted cell suspension across a chip containing thousands of microwells, such that most wells end up with either zero or one cell. Barcoded beads or pre-printed barcoded oligos are then added to each well to tag the transcripts in each cell, similar in principle to the droplet method. The consumables for these systems include the disposable microfluidic chips or well plates and associated reagent kits (barcoded beads/primers, lysis buffers, etc.). A challenge in well-based methods is ensuring random distribution of cells into wells without too many empty wells or double-filled wells – again solved by diluting the cells appropriately. Some chips come in different well sizes to accommodate larger cells or nuclei. Quality of the chips (manufacturing precision and cleanliness) can impact capture efficiency, so sourcing high-quality microfluidic chips is important.
Fluorescence-Activated Cell Sorting (FACS)
For smaller-scale or highly selective experiments, FACS can be used to deposit single cells into the wells of a plate (e.g., a 96-well or 384-well plate) one by one. This approach lets you gate on specific cell properties (using fluorescent markers) to choose which cells to sequence. The consumables here include sorting instruments and sorting buffer, as well as plates compatible with downstream reverse transcription. Each well may contain lysis buffer with a unique barcode (in plate-based indexing methods) or you might later combinatorially index the plates. While FACS gives more control (you can exclude dead cells, doublets, etc. in the sort), it is lower throughput than droplet systems and requires the specialized equipment. It is often used for full-length transcript sequencing methods (like Smart-seq) where cell numbers are lower. If you go this route, Avantor can supply fluorescence-tagged antibodies, cell sorting buffer additives, and sterile PCR plates designed for single-cell collections.
No matter the isolation method, the key challenge is capturing as many single cells as possible without losing them or mixing them. Having the right consumables – high-quality chips/cartridges, calibrated reagents for droplet generation, low-retention tubes for handling cells, etc. – ensures you maximize capture efficiency. Many single-cell capture kits also include reagent additives that support cell lysis and immediate RNA stabilization once the cell is isolated. For example, a droplet reagent kit will have cell lysis buffer and reverse transcriptase mix that gets co-encapsulated, starting the cDNA process right away in each droplet. In summary, choose a capture method that fits your throughput and cell type, and use the recommended single-cell sequencing consumables (chips, oils, cartridges, etc.) to get reliable partitioning of individual cells.
Barcoding and Reverse Transcription: Converting RNA to cDNA
After isolation, each cell (now in its own tiny reaction) is lysed to release its nucleic acids. The next step is to copy the messenger RNA (mRNA) into complementary DNA (cDNA) via reverse transcription (RT). This step is typically combined with adding barcodes to the cDNA. There are two levels of barcoding happening in modern single-cell sequencing kits:
Cell BarcCell Barcodesodes
As introduced above, each cell’s transcripts receive a cell-specific barcode (sometimes called a “cell index”). This barcode is either carried by a bead (in droplet or well systems) or introduced by a primer if using plate-based RT. The result is that all cDNA fragments from cell A share one barcode, and those from cell B share a different barcode, etc., allowing pooled processing of all cells while still being able to separate reads by cell during data analysis.
Unique Molecular Identifiers (UMIs)
In most single-cell RNA-seq library prep kits, each individual mRNA molecule also gets tagged with a random oligonucleotide barcode – a UMI – during the reverse transcription or initial PCR. UMIs label each original RNA molecule uniquely, so that if it gets amplified into many copies later, those copies can be recognized (by having the same UMI) and counted as one original molecule. UMIs help overcome PCR biases, making the quantification of transcripts more accurate. Early single-cell methods suffered from losses and amplification bias, but the introduction of molecular tags has greatly reduced amplification noise and errorsin scRNA-seq.
The reverse transcription stage requires a high-quality reverse transcriptase enzyme that can work efficiently even with tiny amounts of RNA. Many single-cell kits use specialized RT enzymes (often mutated versions with higher processivity and reduced RNAse H activity) to maximize cDNA yield. Additionally, the primers used for RT may be designed for full-length transcripts (e.g., oligo-dT plus a template-switching oligo in Smart-seq chemistry) or for capturing the 3’ end of transcripts (e.g., oligo-dT attached to a cell barcode and UMI in droplet systems). Your choice of single-cell RNA-seq library prep kit will determine the chemistry here: for example, a full-length kit like Takara SMART-Seq v4 will use a template-switching RT to capture entire transcripts, while a 3’ end tagging kit (10x Genomics, Illumina Broad Bio^Rad ddSEQ, etc.) focuses on the tail end of mRNAs. Each has different reagent components.
- Challenges and consumables: One major challenge in this step is reverse transcription efficiency – since capturing every mRNA and converting it to cDNA is impossible, there will be dropout (genes that were expressed in the cell but whose cDNA didn’t get made or amplified). Using optimized reagents can improve capture. For instance, adding trehalose or other additives can stabilize enzymes in droplets; using a primer cocktail that includes both oligo-dT and randomers can help capture non-polyadenylated transcripts or fragmented RNA. Kits often come with these components pre-formulated. Ensure you use fresh reagents here (enzymes not past their freeze-thaw limit, etc.), because the yields are extremely small to begin with. Some protocols also integrate cell lysis buffers that contain detergents and dNTPs such that lysis and reverse transcription happen in one pot – these are provided in single-cell kits to simplify workflow.
To summarize this stage: Barcoding and RT reagents (often supplied as part of a library prep kit) are critical for labeling each cell’s cDNA and generating a faithful representation of that cell’s transcriptome. Investing in a well-validated single-cell RNA-seq library prep kit ensures you have high-quality reverse transcriptase, primer mixes, and buffers that have been tested to maximize cDNA yield from single cells. Avantor’s portfolio includes a range of these kits (for both full-length and 3’-end tagging methods), as well as standalone reverse transcriptase enzymes and PCR reagents suitable for low-input cDNA synthesis if you are developing custom protocols.
cDNA Amplification: Boosting Yield from Low Input
Once reverse transcription is complete, each cell’s cDNA library is still at tiny scale – perhaps yielding only pico-to-nanogram amounts of cDNA per cell. To prepare this material for sequencing, it must be amplified. There are two main approaches to amplify single-cell cDNA:
Polymerase Chain Reaction (PCR)
Many workflows perform PCR amplification of cDNA. In droplet-based methods, for example, after barcoded cDNA is generated in droplets, all droplets are broken to collect the pooled cDNA, which is then PCR amplified in bulk. In plate-based methods like Smart-seq, the cDNA from each cell (now uniquely indexed by well or by a tagmentation barcode) is PCR amplified. PCR amplification requires a robust DNA polymerase. High-fidelity, low-bias PCR enzymes are preferred to maintain representation of different transcripts. Kits often use PCR master mixes optimized for WTA (whole transcriptome amplification) — these mixes might include additives to amplify a broad size range of cDNA and to minimize sequence bias. One example is the KAPA HiFi polymerase or similar high-fidelity enzymes used in 10x Genomics and other kits to amplify cDNA. It’s important to cycle carefully; over-amplification can skew the library toward highly expressed genes. Some protocols include a step to do end-point amplification (stop PCR before hitting plateau) or use quantitative PCR to know when to stop. The consumables here are the PCR enzyme mix, thermal cycler plates/tubes, and cleanup beads (e.g., AMPure beads) to purify amplified cDNA.
In Vitro Transcription (IVT)
An alternative used in certain single-cell methods (like the CEL-Seq family) is to transcribe cDNA back to RNA and amplify via IVT (using T7 RNA polymerase to make many RNA copies, then convert back to cDNA). This method, though less common in high-throughput platforms today, avoids some PCR biases but introduces its own complexities. If your kit uses IVT, you’ll have a kit that provides a T7 transcription mix and reagents for converting amplified RNA back to cDNA.
The main challenge during amplification is maintaining quantitative representation – you want to amplify rare transcripts as efficiently as abundant ones, so you don’t skew the cell’s expression profile. This is why unique molecular identifiers (UMIs) are used: even if bias occurs, UMIs allow bioinformatic correction by counting unique molecules rather than reads. Still, using a high-quality amplification method is important. Ensure your PCR reagents are suited for low-input applications (many vendors label these as “single-cell PCR kits” or “ultra-low input PCR reagents”). These often have higher extension time and more cycles than standard PCR, to get enough yield.
Another consideration is contamination control – because amplification generates lots of product, even tiny contamination can be amplified. Working in a clean area, using nuclease-free tubes and water (Avantor offers molecular biology grade water, PCR-clean plastics, etc.), and including negative control wells will safeguard your results.
Recommended consumables
Look for Whole Transcriptome Amplification kits or amplicon PCR kits optimized for single-cell. These typically bundle the polymerase, dNTPs, primers, and buffers needed. If doing plate-based single-cell (like Smart-seq), you might amplify each cell’s cDNA separately; multi-cell platforms often amplify pooled cDNA together. Either way, PCR enzymes from reliable sources are key. Avantor can supply high-fidelity PCR master mixes, enzyme kits for cDNA amplification, and related cleanup reagents (such as SPRI bead-based cleanup kits to purify amplified DNA). With robust amplification, you’ll turn each cell’s tiny cDNA output into microgram quantities of DNA ready for library prep.
Library Preparation for Sequencing
After obtaining amplified cDNA representing each cell (with barcodes in place), the next step is to create the final sequencing library. In some protocols, the amplification step above already produced the sequencing-ready fragments (for example, in 3’ droplet methods, the cDNA amplification may simultaneously add sequencing adapters). In other cases, you need an explicit library prep step to add sequencing adapters and sample indices (index sequences differentiate different sample pools or batches). Library prep for single-cell libraries is conceptually similar to standard NGS library prep, but must account for the unique structure of the cDNA (with barcodes/UMIs) and the low input.
For full-length methods (like Smart-seq), the amplified cDNA (which is full-length) is often tagmented – i.e., fragmented and tagged with adapters using a transposase (as in Illumina Nextera kits). This is usually done with a modified transposase optimized for small input volumes. Kits such as the Illumina Nextera XT or Nextera Flex are commonly used, or specialized single-cell library kits that perform tagmentation on WTA products. After tagmentation, a short PCR is done to add indices. If you’re using such methods, you will need the appropriate tagmentation-based library prep kit.
For 3’-end methods, often the library fragments already have adapters from the priming stage, so library prep may just involve PCR to append sample indices. For example, the 10x Genomics 3’ gene expression protocol amplifies cDNA with primers that introduce P5/P7 adapters and sample indices. Thus, the “library prep” is integrated with amplification. In those cases, the single-cell library prep kit provided by the vendor covers this process, and your job is mostly to follow the protocol and then perform quality control.
Quality control (QC) at the library stage is extremely important. You should verify the library size distribution (for instance, using an Agilent Bioanalyzer or TapeStation with High Sensitivity DNA chips) and quantify the DNA concentration accurately. Because the libraries are typically complex (many cells worth of data) and will be sequenced deeply, even small errors in quantification can affect sequencing balance. Use nucleic acid quantification reagents that are sensitive enough for diluted libraries – common examples include PicoGreen or Qubit™ dsDNA High Sensitivity assay reagents. These fluorescent quantitation kits (available through Avantor) allow you to precisely measure the ng/μl of your library DNA. Avoid solely relying on spectrophotometers (NanoDrop), as they aren’t sensitive in this range and can be thrown off by residual primers or single-stranded DNA.
If you prepared multiple libraries (say, multiple samples or timepoints), you will index each and then pool them for sequencing. Ensure to use a balanced index set if using combinatorial indices, to prevent index-hopping issues on sequencer. This might involve consumables like an index primer kit.
From a reagent perspective, the library prep step can involve DNA ligases, transposase, PCR kits, and purification beads – often bundled in kits. Choose a single-cell RNA-seq library prep kit that matches your sequencing platform and experiment goal. Avantor provides library construction kits for a variety of applications (RNA-seq, DNA-seq, ATAC-seq, etc.), including low-input kits specifically tested for single-cell applications. These kits simplify adapter ligation or tagmentation and include quality-controlled reagents to maximize your library complexity (representation of as many unique fragments as possible).
By the end of library prep, you should have a sequencing-ready library (or set of libraries) with the following features: each fragment has adapters for the sequencer, each fragment carries a cell barcode and UMI within its sequence, and (if applicable) each library batch has a sample index. Typical yields might be in the nanogram to microgram range of DNA. Confirm the average fragment size (often ~300–600 bp depending on method) and concentration. Now you’re ready to move to sequencing.
Sequencing and Data Analysis Considerations
With libraries in hand, the final experimental step is to sequence them on an NGS instrument (such as Illumina NovaSeq, NextSeq, etc., or other platforms as appropriate). Single-cell gene expression libraries are usually sequenced using paired-end Illumina sequencing, where one read is used to read out the gene fragment and the other read (or a read tag) captures the cell barcode and UMI. Ensure you use a sequencer configuration that provides enough read length to cover the barcode/UMI and the transcript tag — for example, a common format is 28 bp (cell barcode + UMI) and 91 bp (cDNA read) paired-end for 10x Genomics 3’ libraries.
Depth and coverage: Single-cell experiments often generate high numbers of libraries (cells) but with shallow coverage per cell (maybe 20,000–50,000 reads per cell). The exact sequencing depth depends on your application – for basic cell clustering, 20k reads/cell might suffice; for rare transcript detection, you may need more. This means the overall run can be quite large (millions to billions of reads). It’s important to use high-quality sequencing reagents (flow cells and sequencing kits) and to avoid overloading the sequencer beyond recommended cluster densities, as single-cell libraries benefit from uniform representation. Most sequencing consumable kits (flow cell, SBS reagents) will come from the sequencer manufacturer, but Avantor can assist in procuring these and advising on appropriate formats (e.g., mid-output vs high-output kits for Illumina, etc.).
After sequencing, data analysis demultiplexes the reads by their cell barcodes and constructs a gene expression matrix (cells × genes). At this stage, another challenge appears: identifying low-quality cells or artifacts. Cells with very few reads or genes, or those that are likely empty droplets or dying cells, are usually filtered out
Doublets can sometimes be detected bioinformatically if they show expression of markers from two distinct cell types in one cell barcode, although advanced computational tools or multi-omic cross-checks (like transcriptome plus cell-surface protein data) can improve doublet detection. Keep in mind that despite best efforts in wet-lab (dilution, FACS, etc.), you will likely remove a few percent of cells during analysis due to these artifacts. This is normal – plan for extra cells to account for dropouts and doublets.
If you are doing multi-omics (for example, measuring gene expression and cell surface proteins via CITE-seq, or gene expression and chromatin via ATAC+RNA multiome), the sequencing workflow becomes more complex but the principles of good reagent choice remain. You may sequence on multiple read modes (for ATAC-seq, etc.) and then integrate the datasets. It’s crucial to use kits that are compatible with each other – many vendors provide combined multi-omics kits, or you can mix and match (e.g., do RNA-seq and ATAC-seq separately on the same cells sorted into two plates). In any case, performing a multi-omics reagent comparison is wise: ensure that the cell fixation or lysis method needed for one modality won’t ruin the other, and compare available reagent kits for multi-omics to choose one that fits your sample type and throughput. Single-cell multiomics is a fast-evolving field integrating transcriptomic, epigenomic, and proteomic measurements, so new reagent kits are appearing that simplify dual or triple modality workflows. Avantor stays up-to-date with these offerings – from DNA-barcoded antibody reagents for protein detection (e.g., BioLegend TotalSeq™ antibodies) to transposase-based multiome kits – so you can find all the pieces needed for single-cell multi-omics in one place.
Supporting Your Single-Cell Workflow with Avantor
Setting up a successful single-cell sequencing experiment requires coordinating many moving parts: cell processing, specialized reagents, and careful logistics. This is where Avantor can be an invaluable partner. Avantor offers a broad selection of single-cell sequencing consumables covering every step of the workflow – from sample prep to library prep. This includes reagents for cell viability and tissue dissociation, microfluidic chips and droplet generation oils, single-cell RNA-seq library prep kits, high-fidelity PCR enzymes, purification beads, and quality control assays for nucleic acids. By sourcing through Avantor, you gain access to top brands and products in one place, essentially a one-stop shop for building your single-cell pipeline.
In addition to its extensive product catalog, Avantor provides regulated lab support and expertise to help you meet the stringent demands of life science research. Whether you work in an academic lab or a regulated biotech/pharma environment, Avantor’s team can assist with product selection, protocol optimization, and ensuring compliance with any necessary standards. We understand that single-cell experiments often need careful handling (e.g. maintaining cold chains for sensitive enzymes or using certified RNase-free consumables), and our logistics capabilities are up to the task. Avantor’s expertise in logistics and fulfillment means your reagents will arrive on time and in the proper conditions. We manage cold storage and shipping requirements globally, so even the most sensitive multi-omics reagents or sequencing kits reach you reliably. This logistical support minimizes downtime and helps you keep your single-cell project on schedule.
Finally, Avantor is committed to continuous technical support and collaboration. If you have questions about comparing different single-cell kits or need guidance on scaling up your experiment, our specialists are here to help. We can provide insights into multi-omics reagent comparisons, workflow optimizations, and emerging best practices in the single-cell field. Our goal is to empower your research with not only products, but also know-how and service.
Ready to take the next step?
Explore Avantor’s single-cell product collections on our website to find reagents tailored to your needs. From single-cell RNA-seq library prep kits to multi-omics consumables, we have you covered. And don’t hesitate to reach out – speak with an Avantor specialist for personalized guidance on choosing the right reagents for your single-cell sequencing research. With the right kits and support in place, you can confidently launch your single-cell experiments and unlock new discoveries, one cell at a time.