MF-PENS: An Efficient and Versatile Nuclear Extraction Method for Plant Single-Cell Sequencing

Single-cell sequencing is a pivotal technology for advancing plant biology. However, the rigid plant cell wall and interference from cellular metabolites pose substantial technical challenges. To overcome these obstacles, we developed MF-PENS, a novel nuclei isolation method that combines methanol fixation with density gradient centrifugation. Unlike conventional approaches, MF-PENS eliminates the reliance on flow cytometry, preserves nuclear integrity, minimizes RNA leakage, and consistently yields high-quality nuclei suitable for single-nucleus RNA sequencing. MF-PENS has been validated across various plant tissues, including both fresh and frozen rice roots and leaves, as well as challenging sample types such as soybean root nodules, soybean seeds, and tomato fruits. Experimental results demonstrate that MF-PENS effectively improves gene capture efficiency, enhances cell clustering performance, and increases annotation accuracy. With its broad applicability and superior performance, MF-PENS offers a robust and versatile tool for plant single-cell research, deepening our insights into plant development and biology processes.