Biologists might be living through what will one day be known as the “sequencing era” because of the rapid improvements in this technology. For DNA and RNA, advanced instruments make the process faster and the workflow easier to learn. Consequently, the technology can be used by more labs on an expanding range of topics. When asked about the most important recent advance in RNA sequencing (RNA-seq), many experts pointed to single-cell RNA-seq.
With next-generation sequencing (NGS) technology, scientists have used RNA-seq to “detect all RNA transcripts and associated isoforms present in a sample for both RNA discovery and gene-expression analysis,” says Anjali Shah, senior director, product management of clinical next generation sequencing for Thermo Fisher Scientific.
Typical RNA-seq analyzes the RNA from multiple cells as a group. “While this provides very useful information, differences in expression patterns between different types of cells in a tissue or even in culture are not known,” says Senthil Subramanian, a plant biologist at South Dakota State University (Brookings). “Single-cell RNA-seq enables identification of RNA-expression profiles of each cell independently, and this level of detail helps us more accurately define biological processes and make better discoveries.” That’s a pretty big difference in what can be done with RNA-seq.
So, single-cell RNA-seq helps scientists analyze nucleic acids at a very fine scale. In fact, single-cell RNA-seq “enables researchers to characterize the transcriptome on a single-cell level,” says Sameek Roychowdhury, a medical oncologist at The Ohio State University Comprehensive Cancer Center. “This is especially important as we further understand that tissues and organs function as networks of cells that interact in unique microenvironments.”
The wide range of interactions and opportunities in biology for using single-cell RNA-seq promise many discoveries ahead, but it’s all just getting started. “Although single-cell sequencing is still a relatively early-stage market, adoption is quickly accelerating, initially by academic customers addressing a diverse set of discovery applications,” says Gary Schroth—distinguished scientist and vice president, genomic applications department at Illumina. “As the market matures,” he says, “we expect adoption of the tools and techniques by biotech and pharma customers conducting target and biomarker discovery, and diagnostic testing labs seeking higher resolution methodologies to better diagnosis and treat disease.”
Three parts
To sequence the RNA in one cell, it takes a collection of interacting technologies. As Roychowdhury says, “Single-cell RNA-seq is dependent on the efficient isolation of individual cells, high-quality reproducible RNA sequencing for a small input or quantity of RNA, and subsequent bioinformatics analysis to synthesize the data.”
The first step—getting the single cells—can be tricky. “For experiments starting with intact tissue materials, the challenge lies in obtaining individual cells representing all cell types,” Subramanian says. “With recent technological advances, RNA libraries for sequencing can be constructed inside the cells without disrupting them.”
Vendors offer various products that make it easier to get the best starting sample. As an example, Shah says, “Comprehensive sample and library preparation products, such as the Invitrogen RNA purification and Ion Total RNA-Seq v2 kits, deliver increased specificity and sensitivity, wider dynamic range, and simple, strand-specific digital outputs for biological interpretation.”
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In a partnership, Bio-Rad Laboratories and Illumina developed a scalable method for preparing cells for sequencing analysis. “The ddseq system comprises a droplet-based approach,” Schroth says. “This method uses a microfluidic device to compartmentalize droplets containing a single cell, lysis buffer, and a micro-bead covered with barcoded primers.” A cell in a droplet is lysed and released mRNA hybridizes to primer beads. “The droplets are pooled and broken to release the beads,” Schroth explains. “After the beads are isolated, they are reverse-transcribed with template switching.” The resulting cDNAs are amplified with the polymerase chain reaction (PCR), sequencing adapters are added using the Nextera XT Library Preparation Kit, and the barcoded mRNA samples are ready for sequencing.
“Droplet-based approaches enable researchers to analyze cells in a highly parallel manner, at high yield and at attractive costs per cell and experiment,” Schroth says.
The sequencing platform itself doesn’t need to be entirely dedicated to single-cell applications. Scientists can use the same platform that works for whole-tissue or multi-cell RNA-seq, but the technology must work with small amounts of sample. For example, Thermo Fisher Scientific’s Ion AmpliSeq platform requires only 500 picograms of fixed RNA. Schroth points out the Illumina’s “entire sequencing portfolio can be utilized for single-cell sequencing, with best fit depending on the type and scale of the experimental study.”
Depending on the RNA-seq work that a lab requires, other features of a sequencing platform can also be crucial. For example, Shah points out that “Ion AmpliSeq technology allows researchers to amplify thousands of targets in a single tube.” This platform also offers a wide range of reads, 2–260,000,000. In general, different platforms make better fits with different labs. As Schroth says, “The NovaSeq 6000 supports large-scale projects while the NextSeq 550 is ideally suited for studies run in many individual labs.”
Many applications
The number of beneficial ways to use single-cell RNA-seq remains to be seen, but scientists already know that it can be applied in many ways. As one example, Subramanian points out that different types of cells in the human immune system work together to fight off disease. “Within each cell type, there is huge heterogeneity in function—underlying which is a heterogeneity in gene-expression profiles,” Subramanian notes. “The use of single-cell sequencing would enable us to accurately correlate gene-expression profiles with specific functions of each cell.”
The various parts of plants could also be compared. The RNA in cells on the outside of roots, for instance, could be compared to those on the inside. 
“Single-cell RNA-seq enables identifying what happens in different cell types in different scenarios, such as being under stress,” Subramanian explains. “This knowledge helps focus on key targets for manipulation that will help improve plant stress in targeted cells without negatively impacting other cells.”
Image: In this soybean root tip, plant cells with activities of two major plant growth hormones, auxin and cytokinin are tagged green and red, respectively. Cells containing both hormones appear in yellow. These two hormones play a major role in determining the shape and extent of root systems, which help plants obtain nutrients and water from the soil. The image was produced using multiphoton microscopy. Image courtesy of South Dakota State University.
Many other key targets will emerge from basic and applied uses of single-cell RNA-seq. From better crops to safer and more effective drugs, this technology offers many avenues to successful research and development.