Abstract 241: Evaluation of single cell RNA sequencing from FFPE tissue using 10x Genomics Chromium Fixed RNA Profiling
Authors: Black Adam J. , Wiseman Emily , Wang Peng , Wilson Douglas , Torres Fernando , Modarelli Joseph 
Published: April 2023

Abstract
Single cell RNA sequencing (scRNA-seq) uncovers tissue heterogeneity by providing gene expression measurements in individual cells within the tissue. This level of resolution allows for a clearer understanding of specific cellular functions, tissue complexity, and is a powerful tool to discover therapeutic targets and novel biomarkers. A current limitation of scRNA-seq is the requirement to isolate viable cells from fresh or cryopreserved solid tumor biopsies, especially given that formalin-fixed paraffin embedded (FFPE) material are ubiquitously utilized for long-term tissue preservation. Though formalin fixation preserves samples for extended periods of time, it also fragments genetic material, which introduces challenges for many RNA-seq methods due to the reduced size of template material. 10x Genomics recently launched the Chromium Fixed RNA Profiling kit, a probe-based solution for scRNA-seq from fixed cells that enables the detection of &gt;18,000 genes. Herein, this study describes and characterizes the utilization of prepared single cells from FFPE tissue sections to establish sample requirements, key quality control (QC) metrics, and reproducibility of a robust single cell fixed RNA profiling kit for FFPE tissues. To accomplish this, FFPE blocks from breast, lung, ovary, and testes varying in age and quality (i.e., DV200) were sectioned to either 25 or 50 µm for processing. One or two FFPE tissue curls per sample were dissociated into single cell suspensions using the Miltenyi FFPE Tissue Dissociation Kit, processed through the Chromium Fixed RNA Profiling workflow, sequenced using the Illumina NovaSeq, and analyzed by Cell Ranger 7.0.1. All samples yielded sufficient cells for probe hybridization and for targeting the capture of 10,000 cells. The total number of genes detected was highly reproducible between replicates and ranged between 14,000 and 18,000 per tissue type, indicating robustness of the workflow across tissues. Notably, DV200 scores were observed to strongly correlate with key QC metrics, specifically cell capture efficiency and RNA species diversity. While FFPE curl number and size input variables were found to have no significant impact on QC metrics, the effect of DV200 was observed within each tissue type, indicating a strong driver of metric variability. These combined findings support the use of the 10x Genomics Fixed RNA Profiling workflow to obtain high quality single cell gene expression data from FFPE tissues. These data also demonstrate that meaningful gene expression data is produced by multiple tissue types and input amounts, but samples with the highest DV200 produce the most robust data. Workflow optimizations focused on reduction of required tissue input to maintain reproducible cell population characterizations will be crucial for facilitating the interrogation of tumor FFPE samples with limited material availability.
Citation Format: Adam J. Black, Emily Wiseman, Peng Wang, Douglas Wilson, Fernando Torres, Joseph Modarelli. Evaluation of single cell RNA sequencing from FFPE tissue using 10x Genomics Chromium Fixed RNA Profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 241.