MF-PENS: an efficient and versatile nuclear extraction method for plant single-cell sequencing

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Abstract The widespread adoption of single-cell transcriptomics in plants remains constrained by the challenges of isolating high-quality nuclei from tissues with rigid cell walls and complex metabolomes. Here, we present m ethanol f ixation and P ercoll- e nriched n uclei s orting (MF-PENS), a robust strategy designed to tackle these limitations. Distinct from conventional protocols, MF-PENS circumvents the need for fluorescence activated cell sorting (FACS) and effectively reduce RNA leakage, consistently yielding high-quality single-nucleus RNA sequencing (snRNA-seq) data. MF-PENS has been validated across various plant tissues, including fresh and frozen rice roots and leaves, as well as recalcitrant samples such as soybean root nodules, soybean seeds, and tomato fruits. Benchmarking results demonstrate that MF-PENS significantly enhances gene capture efficiency, cell clustering performance, and cell type annotation accuracy. With its broad applicability and superior performance, MF-PENS represents a robust and versatile tool for plant single-cell research, facilitating deeper insights into plant development and biology processes.

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