Performance of the 10X Genomics flex single-cell sequencing assay and its application to overcome challenges in clinical trial samples

Single-cell RNA sequencing (scRNA-seq) has become essential for understanding disease biology, yet its application in clinical trials is often limited by logistical challenges associated with the handling of biospecimens. Newly developed protocols aim to address these limitations by enabling profiling of fixed tissue. These new solutions need to be benchmarked against well-established protocols to assess their performance and suitability for clinical research. In the present study we systematically compared different fixed-tissue and nuclei-based RNA-sequencing workflows in a set of solid tumor samples, which were collected and preserved following common procedures in clinical trials. When applied to both frozen tumor tissue and FFPE blocks, the 10X Genomics GEM-X Flex Gene Expression assay, in combination with the “chop-fix” preprocessing protocol, demonstrated enhanced data quality—characterized by higher gene and UMI detection sensitivity and slightly reduced ambient RNA background—relative to the standard GEM-X Universal Gene Expression solution. Furthermore, this protocol improved the capture of specific biological signals and resolved cell states that were less distinct with conventional approaches. GEM-X Flex generates reliable, comprehensive transcriptomic data from both fixed tissue and clinical biopsies. By overcoming some of the limitations of fresh and frozen tissue analysis, this protocol offers a robust solution for the broad implementation of scRNA-seq in clinical trials.