Cross-species optimization of nuclei isolation in ten plant species

Single-cell technologies are transforming plant biology, yet broadly transferable nuclei isolation remains a key bottleneck for snRNA-seq. We developed a reproducible, cost-efficient Percoll-based workflow that is applicable to multiple maize tissues and nine additional plant species. In maize, nuclei from root, shoot, leaf, and embryo consistently concentrated at the 80% Percoll interface and exhibited high integrity, with typical recoveries > 50,000 nuclei per sample. For other species, gradient compositions were tuned according to genome size to achieve efficient enrichment and clean suspensions, and yields ranged from 17,000 to 40,000 nuclei per sample. Downstream validation showed that nuclei from special interest maize and Tripsacum generated high-quality snRNA-seq libraries, as supported by cDNA quality profiles. These results demonstrate the versatility and robustness of the method across species and tissues.