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

Single-cell sequencing is a crucial tool for exploring plant biology, yet the rigidity of plant cell walls and metabolite interference pose significant experimental challenges. To address this, we developed a novel nuclear preparation method, MF-PENS, which integrates methanol fixation with density gradient centrifugation. This method eliminates the need for flow cytometry, ensures nuclear integrity, prevents RNA leakage, and provides high-quality data for single-nucleus RNA sequencing (snRNA-seq) analysis. MF-PENS has been validated across various plant tissues, including fresh and frozen rice roots and leaves, as well as complex tissues such as soybean root nodules, soybean seeds, and tomato fruits. Experimental results demonstrate that MF-PENS effectively enhances the gene capture efficiency and cell clustering accuracy of snRNA-seq, providing reliable data support for downstream analysis. The method’s broad compatibility and high efficiency provide a powerful impetus for plant single-cell research, advancing a deeper understanding of plant biology and developmental mechanisms.