PIPsceq: powering single cell RNA sequencing into the million cell era

Project Summary/Abstract The dysregulation of multicellular regulatory networks is associated with nearly every human disease including cancer, aging, autoimmunity and neurodegeneration. These networks of chemical, mechanical and electrical signals allow cells organized within tissues to regulate collective decisions and behaviors. Our understanding of the cell-cell interactions that underlie this dysregulation is hampered by a lack of tools to enable interrogation of biological networks at the single cell level. In this Phase I SBIR, we will develop a generalized platform for quantitative measurement of transcriptional changes of single cells at the massive scale required for highly complex mechanistic studies of human disease. The routine availability of high-throughput tools for single-cell RNA transcriptional analysis (scRNAseq), such as those based upon droplet microfluidics, has enabled new mechanistic studies of cell types across tissues. These tools are based upon loading of individual cells into water-in-oil droplets at limiting concentrations. However, these technologies have important limitations prohibitive for highly complex mechanistic studies including lack of throughput necessary to efficiently analyze hundreds of samples simultaneously or isolate andgt;10,000 cells in a given reaction, high costs associated with microfluidic devices and consumables, and problematic artifacts (e.g. doublets and batch effects). Recently, scRNAseq sample multiplexing techniques have been described that introduce an oligonucleotide barcode to mark all of the cells in a given sample such as the MULTI-seq approached developed by our collaborators Zev Gartner and Eric Chow. We propose to expand the capability of Fluent BioSciences patented self-assembly technology, Pre-templated Instant Partitions single cell expression quantitation (PIPsceq), for sample multiplexing applications to enable high-complexity perturbational studies. PIPsceq is a controlled liquid emulsification technology capable of instantaneous partitioning of millions of droplets without the use of microfluidics representing up to 1000X increase in cell throughput per reaction over existing droplet technologies. The sample multiplexing application (e.g. MULTI-seq) for PIPseq will form the basis of a generalized platform for mechanistic studies of human disease by enabling paradigm-shifting increases in the scale of digital droplet biology as well as reduce sample preparation costs and artifacts. Our overarching goal is to develop prototype PIPsceq library preparation kits for sample multiplexing applications for andgt; 100,000 cells per reaction, which we believe will enable highly complex perturbational studies of individual cells that are not feasible with existing technology. The aims of this Phase I SBIR are to demonstrate cell capture of an unprecedented 10 million cells using PIPsceq and to produce prototype PIPsceq 100,000 cell library preparation kits to be used for performance benchmarking against commercial and published academic scRNAseq technologies.Project Narrative The dysregulation of multicellular regulatory networks is associated with nearly every human disease including cancer, aging, autoimmunity and neurodegeneration. Fluent BioSciences is developing new tools that enable high resolution analysis of individual cells which we believe will form a generalized platform for mechanistic studies of human disease. This technology relies on the core innovation of our company for performing single cell genomics using a bulk self-assembly process that requires no microfluidics or specialized equipment.