PROJECT SUMMARY / ABSTRACT Single cell RNA sequencing (scRNAseq) provides unprecedented insight into the gene regulatory pathways of living cells in complex tissues. Current single cell technologies can provide high-quality, high resolution analysis of perturbations in cellular populations, but are limited in terms of access, affordability, and scalability of cell input. In order to broadly deploy scRNAseq as a high-throughput, cost effective research and discovery tool, a platform requires several characteristics: sensitivity to detect subtle changes in gene expression as a result of pharmaceutical intervention, scalability in order to efficiently process hundreds to hundreds of thousands of input cells, a protocol capable of processing multiple control and treatment conditions, and efficient multiplexed sequencing capability for cost-effectiveness. Fluent BioSciences has developed Pre-templated Instant Partitions for single cell RNA sequencing (PIPseq) as a novel approach toward scRNAseq that has key advantages over existing methods. PIPseq provides unprecedented accessibility to scRNAseq for applications that are not readily addressable by competing technologies. Eliminating the dependence on complex instrumentation and expensive microfluidic consumables allows even modestly equipped cell biology labs to implement powerful scRNAseq assays. The intrinsic scalability of the PIPseq platform is also particularly useful in applications where many patient samples or treatment conditions might be evaluated. This direct-to-phase II proposal will establish commercial-readiness of PIPseq kits through rigorous internal stability and reproducibility testing, external evaluation and reproducibility testing with multiple genomics core laboratory collaborators, and pre-clinical evaluation of primary breast tissue samples provided by an academic collaborator.