Abstract 1440: GEM-X Universal Multiplex: a cost-effective, sample agnostic multiplexing solution for scRNA-seq in cancer research

Abstract Resolving heterogeneity within cancer subtypes is critical to better understand the underlying disease mechanisms and to select the most effective treatment options. Single cell RNA sequencing (scRNA-seq) has revolutionized cancer research by providing unprecedented insights into tumor heterogeneity, immune microenvironment, and treatment responses - areas that were previously difficult to explore using bulk RNA-seq. Recently, sample multiplexing has emerged as a significant advancement in scRNA-seq workflows, enhancing throughput, reducing library preparation workload, and lowering per-sample costs. However, traditional multiplexing methods usually require either samples to have distinct genotypes, or the application of cell-specific barcoding before sample pooling, leading to challenges such as high cell input requirements, laborious sample preparation steps, and compatibility limitations across species and sample types. To address these issues, we present GEM-X Universal Multiplex, a cost-effective, streamlined approach that eliminates the need for upstream tagging, and offers greater flexibility across different sample types, species, and assays. GEM-X Universal Multiplex utilizes a microfluidic chip to co-partition cells from up to four samples, each with a unique gel bead set, simplifying the multiplexing process. After encapsulation, library generation, and sequencing, 10x Barcodes are employed to trace cDNA-derived reads back to their original sample and gel bead set. We validated GEM-X Universal Multiplex using human kidney dissociated tumor cells derived from a renal cell carcinoma patient and fixed peripheral blood mononuclear cells from patients with acute lymphoblastic leukemia, primary myelofibrosis, multiple myeloma, and squamous cell carcinoma. Our platform demonstrated its ability to uncover the heterogeneity of different sample types and their unique clonotypes. We also compared GEM-X Universal Multiplex with our singleplex product, GEM-X Universal. Both approaches yielded comparable data in terms of library quality and complexity, the number of V(D)J clonotypes detected, and consistent cell type identification and proportions. In conclusion, GEM-X Universal Multiplex provides a powerful and scalable tool for cancer research, offers high accuracy and reduced technical challenges, and enables broader applications in the study of cancers. Citation Format: Jui-Szu Chen, Sunny Yang, Joe Shuga, Winnia Hui, Ace Santiago, Ivan Akhremichev, Brett Olsen, Daniel Reyes, Mohammad Rahimi, Peter Smibert, Jessica Terry. GEM-X Universal Multiplex: a cost-effective, sample agnostic multiplexing solution for scRNA-seq in cancer research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1440.