- Sony Biotechnology
- Sony Biotechnology
Single-cell sequencing has proven invaluable to understanding the biology of heterogeneous samples such as brain or tumor tissues.
"Cells are often quite plastic," explains Andrew Adey, PhD, Assistant Professor of Genetics at the Oregon Health and Science University in Portland. "Cells within a tumor, for example, can be in one epigenetic state, but when you treat them with a drug, they can shift states. We're interested in what configurations and properties of a cell identify it as a specific type, lock it in one developmental state or push it to the next, or make it resistant to that drug."
"The Sony SH800 [is] the major workhorse of our workflow."
To address these questions, Dr. Adey's lab has developed a new methodology called single-cell combinatorial indexing (SCI).1 "SCI is a way of generating high-throughput, scalable numbers of single cells in library preparation for a variety of different properties without the need for specialized equipment," he says. "All you need is a cell sorter. In our case, we're using the Sony SH800, and that's the major workhorse of our workflow."
Although SCI is designed to analyze properties of single cells, Dr. Adey's lab never sorts single cells into individual wells. Instead, they label cells with multiple DNA "barcodes" and distribute them into place using the SH800 sorter. The workflow is shown in Figure 1. "Once the barcoded nuclei are sorted into the new wells of a plate, we can add a second barcode and get single-cell information without ever handling single cells, in a much higher throughput than the actual number of wells that we're sorting. Instead of one cell library per PCR well, we're able to get tens to hundreds of single-cell libraries per PCR well. It dramatically reduces costs and increases throughput."
Dr. Adey's lab has shown that SCI can be applied to study a variety of epigenetic properties such as chromatin accessibility2 and DNA methylation.3 "Our interests center around gene regulation and how cell types are established and maintained, as well as the plasticity and dynamic properties of different cells and cell types," he says. "With dynamic cells that are differentiating or shifting their epigenetic state in response to a drug, we can get high-precision maps of the ordering and specific events that occur during that state transition. Those concepts are applicable to broadly different areas of biology. About half my lab works in the cancer space and the other half in the neuroscience space. In both areas, single-cell 'omics'—studying cellular genomics, epigenomics, and transcriptomics at the single-cell level—have proved extremely valuable."
The core of the SCI method is fluorescence assisted nuclei sorting (FANS), in which the nuclei (rather than whole cells) are isolated and sorted based on DNA staining with DAPI. The lab uses the SH800 from Sony to address the multiple challenges this process presents. "We do a lot of molecular gymnastics to get barcodes where they need to be on these sequence molecules and get everything in the right format," explains Dr. Adey. "We often perform chemical and enzymatic reactions while the nuclei are still intact. To get genome-wide signal, we need to remove nucleosomes. But as soon as you do that, the DNA turns into a 'spring' that explodes the nucleus, so that when we sort, it's all just debris! When we see that much debris on the SH800, we know something went wrong upstream. We've developed workflows and chemistry to process the nuclei and gate out normal debris, so that we get a clean single-nucleus gate that we can sort on and deposit those barcoded nuclei."
A second challenge is that nuclei are "sticky" and tend to clump together. "Isolating the singlet population is critical," Dr. Adey says, "because doublets might result in barcode collisions and confound the results. This is a big challenge in primary cancer samples because tumor cells often have higher ploidy. We really need to distinguish between mitotic cells, stromal cell doublets, and cancer cells. We have developed a variety of sorting parameters on the Sony SH800 to accomplish that. It has the resolution and capability to clearly isolate single nuclei using differences in DAPI peaks and light scatter."
"When someone new comes into the lab, we have them shadow an experienced user through the protocol on the SH800 and then do it themselves. They can then do it on their own the next day. That's impressive!"
When Dr. Adey arrived at OHSU, a neighboring lab offered to share their SH800. "They used it a few times a month," he laughs. "We use it almost every day!" He was especially pleased with the SH800's ability to sort nuclei compared with other sorters. "From the start, the gating worked really well. We were so happy with the SH800's ability to sort DAPI-stained nuclei that we purchased our own, which arrived in December. We optimized the new instrument specifically for high-throughput DAPI nuclei sorting, so we're quite excited about it."
A second advantage was that the SH800 was intuitive to use. "Many individuals in my lab need to be able to operate the instrument, because they all do their own experiments and preps. When someone new comes into the lab, we have them shadow an experienced user through the protocol on the SH800 and then do it themselves. They can then do it on their own the next day. That's impressive! Back when I was at the bench sorting, I was never comfortable enough to run a sorter myself. A short training period is important because we use it for so many different projects."
Dr. Adey also appreciates the SH800's disposable fluidics chip. "We switch between human and mouse samples frequently with different projects," he explains. "If we're looking at human cells and see contamination from mouse cells, that would be a problem. But we don't detect any of that, probably due to using a different chip for each experiment."
"An easy-to-use sorter is like a PCR machine—a standard piece of equipment that you can train on quickly and not worry about that step in the protocol."
For Dr. Adey, having a cell sorter right in his lab is critical to their multistep workflow. "We love our core facility. But we often do sorting on one step, and then we have a one- to four-hour gap where we're processing, and then we need to sort again. Scheduling a core sorter for those blocks of time—especially when someone in the core has to actually run the sample—can be a challenge. Maybe a sample from the clinic comes in at noon, which doesn't sound that late, but when you've got a long protocol ahead of you and you rely on help from a core with a tight schedule, that can be a problem. The volume of sorting we do and the flexibility it requires would be impossible on a widely shared instrument such as [in] a core facility. An easy-to-use sorter is like a PCR machine—a standard piece of equipment that you can train on quickly and not worry about that step in the protocol."
Dr. Adey, who received his PhD in 2014, has already won an Early-Career Award from the American Society of Human Genetics (ASHG), and his research has been featured in the blog of Dr. Francis Collins, director of the National Institutes of Health. What's the secret to his productivity? "I've been very fortunate to have incredible mentors and advisors, support from leadership here at OHSU, an excellent team of hardworking and talented individuals, and some excellent collaborators here. Also, not working too hard! I encourage everyone in my lab to have something that keeps them sane outside the lab."
And what is Dr. Adey's favored recreation? Homebrewing. (He lives in Portland, after all.) "But there's a lot of science in it too. I'm even considering sequencing some yeast strains," he laughs. "It's all in good fun."
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