Genetic Engineering & Biotechnology News

OCT15 2017

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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14 | OCTOBER 15, 2017 | | Genetic Engineering & Biotechnology News Nanodrop RNA-Seq Earlier methods examining gene expression usually aver- aged all the transcriptomes obtained from bulk tissue. This global snapshot necessarily lost vital information at the single-cell level. While recent advances enabled mRNA-seq analysis of individual cells, technologies could only examine a few hundred cells at a time. Additionally, cells often first needed separating by flow cytometry or microfluidics. However, the emergence of high-throughput single-cell RNA-seq (scRNA-seq) has begun revolutionizing the field. "This technology has offered an exciting, cutting-edge tech- nique that provides unprecedented insight into the expres- sion patterns of thousands of single cells," reports Muriel Breteau, Ph.D., technical applications specialist, Dolomite Bio. She adds, "Individually analyzing thousands of single cells from a tissue can vastly improve the [understanding of] complexity of biological systems to derive vital information [about] diseases and immunity." Dolomite Bio has developed a microfluidics platform that enables the Drop-seq protocol (Figure 1). Dr. Breteau explains, "Our technology allows highly parallel genome- expression profiling of cells using nanoliter droplets. Thou- sands of cells can be individually captured in these droplets. Inside the droplet, and within only a few minutes, the indi- vidual cell is lysed and its mRNA captured on uniquely bar- coded oligonucleotides attached to beads. After recovering the beads, they are subjected to reverse transcription, library preparation, and finally sequencing" (Figure 2). As an example, a cell suspension can be loaded into an agitated remote reservoir and beads injected. Droplets are then collected in an output reservoir at ~2,800 droplets per second. Within a 15 minute run, more than 6,000 single-cell libraries can be generated. Dr. Breteau points out, "There are many applications for this novel technology ranging from fundamental research identifying new cell types, to defining tumor heterogeneity, clonal evolution, and effects of drug treatments in oncology applications. One of the key features of the system is that it is open and flexible. Researchers can innovate by utilizing their own reagents and protocols instead of being required to use just [Dolomite Bio's products]." In the future, Dr. Breteau envisions the day when the technology will be applied to personalized medicine. "Be- cause this is essentially a lab-on-a-chip microfluidics device, it could be possible to obtain a small blood sample and get quick answers, for example, as to the very early stages of cancer detection." OMICS Feature See Single-Cell Genomics on page 18 The Rise of Single-Cell Genomics Continued from page 1 Figure 1. Dolomite Bio has developed a process that uses nanoliter droplets to encapsulate individual cells along with mRNA capture beads (circles with squiggly lines). The process, which enables high-throughput single-cell RNA sequencing, consists of the following steps: Separate suspensions of cells and beads are combined before being dropletized in oil on a microfluidic chip. Inside a given droplet, an individual cell is lysed and its mRNA is captured on a single, uniquely barcoded bead. The droplets are broken, and the mRNA-coated beads are recovered. Subsequent reverse transcription produces bead-bound single-cell libraries that are uniquely barcoded by the cell of origin. Figure 2. Muriel Breteau, Ph.D., a technology applications specialist at Dolomite Bio, injects barcoded oligonucleotide- coated beads into a single-cell RNA-sequencing system. The system, which incorporates the company's scRNA-Seq chip, captures mRNA from lysed cells. Once the mRNA is encapsulated in nanoliter droplets, genome-wide expression profiling at the single-cell level may commence. Figure 3. The BD Rhapsody, from BD Genomics, can enable digital quantitation of hundreds of expressed genes across tens of thousands of single cells in parallel. To accomplish single- cell capture and molecular indexing of mRNA transcripts, BD Rhapsody uses magnetic oligonucleotide barcoded beads. These beads are then pooled into a single tube for cDNA amplification and library construction. BD Genomics says that users will be able to multiplex many samples in one run with a single-cell multiplexing kit, significantly increasing sample throughput while maintaining the low doublet rate. " This is the first look at the entire transcriptional profile in individual neurons activated by external stimuli and is a critical step in ultimately discovering how a memory is captured and stored." –Dr. Lasken

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