SAN FRANCISCO (GenomeWeb) – University of Washington spinout Split Biosciences is commercializing a single-cell RNA sequencing technology that it aims to launch through an early-access program this summer.
The technology relies on fixing and immobilizing single cells, rather than physically isolating them, in order to do single-cell RNA sequencing. The startup, which currently has four full-time employees, raised around $1.2 million in seed financing last year.
The UW researchers first described the technology last year in a publication in Science and also at this year's Advances in Genome Biology and Technology meeting in Marco Island, Florida.
Following the Science publication, the researchers won a $150,000 grant from the Washington Research Foundation to optimize the technology and develop a kit, which they then tested with researchers from Seattle Children's Hospital, the Fred Hutchinson Cancer Research Institute, and others, said Georg Seelig, a Split Bio cofounder and its CSO. After that, the team, which has filed patents on the method through the university, spun out Split Bio.
The method, dubbed SPLiT-seq for split-pool ligation-based transcriptome sequencing, relies on using a single cell itself as a compartment. Cells are formaldehyde-fixed, which essentially freezes their activity, so the gene expression profile is the same as when the cell was extracted from the tissue, Seelig explained, and the RNA molecules stay within the cell. Fixation also enables researchers to store samples before analyzing them. After fixing the cell, the cell membrane is made permeable by adding a detergent that essentially "pokes holes" into the membranes so that reagents can be added directly to the cell, said Split Bio CEO and cofounder Alex Rosenberg. These steps are standard and common lab processes.
The cells are then dispersed into a 96-well plate where barcoded reverse transcriptase primers are added to create cDNA within the cell itself. The cells are then pooled and redistributed and well-specific barcodes are added to the cDNA molecules within each cell. The process is repeated, and in the third round of barcoding, a unique molecular identifier is also added. In a fourth and final round of barcoding, the cells are also divided into sub-libraries with sequencing barcodes.
An advantage of the method, said Charlie Roco, the company's chief technology officer and a cofounder, is that it does not require a separate instrument. "So, it's accessible to any lab," he said.
Split Bio's barcoding strategy is also similar to combinatorial indexing methods used for single-cell sequencing as described by Jay Shendure's group, also at UW.
Seelig said that the two methods were developed independently at UW and while "conceptually similar" in that they both use combinatorial barcoding, many of the details, like the specific mechanisms for barcoding and the cell treatments, are different.
In the Science study, the researchers sought to determine the performance of the method, ensuring that it truly yielded results from single cells, by analyzing mixtures of human and mouse cells. When they analyzed 1,758 uniquely barcoded cells, the researchers found that 99.9 percent could be assigned to a single species. When scaled up to process around 100,000 cells, the doublet rate would be about 1 percent, Seelig noted.
Since the publication, the team has continued to optimize the protocol. For instance, it has "reduced the number of steps required and optimized reaction buffers to increase sensitivity and minimize hands-on time," he said.
In addition, the researchers have internally compared the method to other tried-and-true single-cell protocols like Smart-seq, Drop-seq, and 10x Genomics' single-cell methods and found that the results are comparable. Importantly, Seelig said, there seems to be little method-based bias and the results cluster by cell type, rather than by the protocol used.
The team has also collaborated with the Allen Institute for Cell Science to use the method to study the differentiation of induced pluripotent stem cells to cardiomyocytes. For that collaboration, the group collected around 48 samples over 90 days, fixing and freezing them, and then ran the experiment at the end. Seelig said that the team plans to publish results from its external collaborations and internal research in the future.
Going forward, the firm plans to develop other applications, he said, including combining single-cell RNA-seq of T cells with their receptors, applications for microbiome sequencing, as well as versions that are compatible with formalin-fixed paraffin-embedded samples. In addition, it plans to work on techniques that incorporate spatial information.