The Method Man

How Will Greenleaf, ATAC-seq and C1 Open App are pioneering single-cell epigenetics

The draw of single-cell analysis lies in scientists’ desire to understand how we, as biological systems, work. Discovery upon discovery leads to deeper questions about passive versus causal mutations, transcription mechanisms, pathway regulation, cellular function. It can get really complex, or begin to sound misleadingly simple:

“Where’s the brain of the cell?”

Will Greenleaf takes a breath. His passion for his work is clear as he continues talking: He’s quick yet thorough, making sure to complete each idea before moving on at a pace that suggests the next thought is running ahead of him.

Head of the Greenleaf lab and assistant professor of genetics at Stanford, Will Greenleaf, PhD, is pioneering single-cell research in epigenetics, the study of cellular trait variations not caused by changes in the DNA structure. It’s a field very likely to benefit from single-cell’s ability to dramatically enhance understanding of cell structure and behavior, and Greenleaf is leading the effort with publication of a new method called single-cell assay for transposase-accessible chromatin using sequencing, or scATAC-seq. His findings were published in Nature in June, 2015, and the method is now downloadable from Script Hub™ for use with any C1™.

The Greenleaf lab focuses on understanding the physical genome, how the DNA material is actually compacted into each cell and how different segments of that DNA are used. “If I stretched your genome out from a single cell in your body,” Greenleaf explains, “it would be two meters long and two-billionths of a meter wide. And that gets folded and compacted into a nucleus that is five-millionths of a meter in diameter.” He likens it to taking a telephone cord the length of the United States and packing it into a standard two-story house.

“It’s an incredibly complicated problem,”

Greenleaf continues. “You’ve got all this stuff, encoding stuff, much of which, depending on the cells in your body, is sort of irrelevant. In a given cell, maybe two or three percent of the genome is actually out of the boxes.”

That unboxed bit of the genome is the “accessible” DNA that ATAC-seq was created to identify. The assay binds only to the accessible DNA regions, adding a nucleic acid tag that enables sequencing. Epigenetics is a very young field in biology, and new hypotheses indicate that cell-to-cell variability in gene expression is caused by changes in chromatin state. Studying the accessible chromatin at the single-cell level can reveal the source of cellular heterogeneity. Once the accessible regions are tagged, high-throughput sequencing lets scientists map them back to the genome to understand which parts are used by that particular cell.

It makes sense that this is Greenleaf’s focus. A student of physics, chemistry and even computer science, he found a career that reflects a desire to understand how things work at their base level. Much of his study has involved adopting and developing means to help further that understanding. “Ideally we develop tools and then apply them to interesting and fundamental questions—previously unanswerable questions—in biology,” he says. “My vision for the lab is mixing these new techniques with new biological insights.”

His interdisciplinary, pioneering approach served him well as a student at Stanford, where he benefited from access to leading minds in various fields. Coming from what he calls “a naturally collaborative atmosphere,” Greenleaf is innately inclined to work with diverse partners to advance research possibilities. For single-cell ATAC-seq, he worked closely with Howard Chang and his lab at Stanford, and he credits Jason Buenrostro, a co-author of the paper and senior graduate student in both Greenleaf and Chang’s labs, with really driving the work forward. Other essential collaborators include Fluidigm Senior Scientist Dave Ruff and Product Application Specialist Michael Gonzales, as well as the product that made it all possible: Script Builder™. Part of the C1 Open App™, Script Builder allows scientists to develop new methods for single-cell analysis.

“It just makes a lot of sense for people with specific areas of expertise to get together and extend science right at the edge of where the disciplines merge,” Greenleaf says. Advancements in sequencing and analysis have made biological research more complex than ever, he notes. “That makes the community aspect all the more important.”

The C1 Open App is comprised of Script Hub and Script Builder, which work with all existing C1 systems. Users can visit Script Hub to view and download original scripts for new methods to run on C1, including ATAC-seq. Script Builder is an open program that enables users like Greenleaf to develop their own research and analysis methods.

“Script Builder definitely opens the door for playing around with custom methods,” Greenleaf says, “to use what is a very powerful, generalizable platform for doing single-cell analysis—the C1 instrument itself —and repurpose it to do custom assays.

“I think there’s been a push in scientific publishing toward transparency and the ability to reproduce results,” he continues. “It’s all the better if we can get closer to a platform that allows the experimental side to be easily reproduced by others, because that’s the whole point of the scientific endeavor, right? To allow other people to either reproduce results or extend work to other systems.”

The approach reflects a recent trend in biological research of merging nontraditional fields, DIY science and crowdsourcing. As the biology community discovers more about how systems work, it often requires more input and insight from outside fields of study, including statistics and computer science. There’s a sense that everyone needs to work together to find answers.

Greenleaf’s tendency toward collaboration extends to his conversational style. He speaks with the confidence of someone at the leading edge of his field, but in an open, participatory, even leading manner, as if the talk itself is an opportunity for him to discover something new. When asked what inspires him, Greenleaf pauses for the first time, considering answers to a question he’s never asked.

“I get inspired by the work of a lot of really visionary scientists who are making incredible contributions to the genomics field,” he says finally, reluctant to name names in case he forgets one. “All of my work stands on the shoulders of the people who have pioneered those sorts of approaches.”

Now with single-cell ATAC-seq, Greenleaf stands solidly as one of those pioneers, ready for others to step up.


This blog was written for Fluidigm Corporation, a biotechnology and life sciences firm based in San Francisco. View the original here.

Leave a Reply

Fill in your details below or click an icon to log in: Logo

You are commenting using your account. Log Out /  Change )

Twitter picture

You are commenting using your Twitter account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s