Abstract 2781: Enabling low cell input and high throughput processing of cell fixation for single cell RNA profiling using combinatorial barcoding

Abstract Single cell analysis is a critical approach to tease out nuanced information in any biological system. Combinatorial barcoding is an extremely efficient method for scRNA-seq at scale, achieving both sensitivity in gene detection and flexibility in sample input. This approach, however, is challenging when starting with sample inputs of less than 100,000 cells. To address this, we have developed a modified fixation and cell capture method for low sample inputs that is compatible with the combinatorial barcoding approach. This allows researchers working with low sample inputs to fix, store and barcode their cells efficiently without the need for costly or specialized instruments. Low Input Fixation can be used to fix and profile up to 384 samples in parallel, results in a shorter workflow, and enables consistent handling (which minimizes both experimental error and burden on scaling). This method maintains the stability of fixed samples at -80°C and capture efficiency remains consistently high for inputs as low as 10,000 cells. The workflow maximizes sample recovery while providing accurate and comprehensive results of transcriptional changes from experimental perturbations. As proof-of-concept we screened a cancer drug panel with cell input amounts between 10,000 - 20,000 cells per treatment. Robust cell capture minimizes loss during drug perturbations resulting in consistent gene detection. Whole transcriptome analysis using Evercode WTK identifies changes in gene expression associated with each drug class and cell type. Results of the screen validate putative drug targets in the panel. Additionally, panel-based perturbation allows for detection of off-target impacts at scale. This work demonstrates the combination of low input fixation with combinatorial barcoding to enable high throughput cancer drug and compound screening. Citation Format: Olivia Kaplan, Sarah Schroder, Vuong Tran, Simone Marrujo, Alec Salvino, Ajay Sapre, Ian Dowsett, Zeynep Sayar, Alex Koriath, Alex Sova, Gary Geiss, Charles Roco, Alex Rosenberg. Enabling low cell input and high throughput processing of cell fixation for single cell RNA profiling using combinatorial barcoding [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 2781.