FX-Cell: a method for single-cell RNA sequencing on difficult-to-digest and cryopreserved plant samples

Single-cell RNA sequencing in plants requires the isolation of high-quality protoplasts-cells devoid of cell walls. However, many plant tissues and organs are resistant to enzymatic digestion, posing a significant barrier to advancing single-cell multi-omics in plant research. Furthermore, for field-grown crops, the lack of immediate laboratory facilities presents another major challenge for timely protoplast preparation. Here, to address these limitations, we developed FX-Cell and its derivatives, FXcryo-Cell and cryoFX-Cell, to enable single-cell RNA sequencing with both difficult-to-digest and cryopreserved plant samples. By optimizing the fixation buffer and minimizing RNA degradation, our approach ensures efficient cell wall digestion at high temperatures while maintaining high-quality single cells, even after long-term storage at -80 °C, and circumvents use of nuclei, which are not representative of the pool of translatable messenger RNAs. We successfully constructed high-quality cell atlases for rice tiller nodes, rhizomes of wild rice and maize crown roots grown under field conditions. Moreover, these methods enable the accurate reconstruction of plant acute wounding responses at single-cell resolution. Collectively, these advancements expand the applicability of plant single-cell genomics across a wider range of species and tissues, paving the way for comprehensive Plant Cell Atlases for plant species.