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FX-Cell: a method for single-cell RNA sequencing on difficult-to-digest and cryopreserved plant samples
Xin Ming1, Mu-Chun Wan1,2, Zheng-Da Zhang1
1Key Laboratory of Plant Carbon Capture, CAS Center for Excellence in Molecular Plant Sciences (CEMPS), Institute of Plant Physiology and Ecology (SIPPE), Chinese Academy of Sciences (CAS), Shanghai, China.
Nature Methods
|November 27, 2025
Summary
New FX-Cell methods enable high-quality single-cell RNA sequencing for challenging plant samples, including cryopreserved tissues and difficult-to-digest organs. This advances plant genomics and the creation of comprehensive plant cell atlases.
Area of Science:
- Plant Biology
- Genomics
- Molecular Biology
Background:
- Single-cell RNA sequencing (scRNA-seq) in plants is crucial for understanding cellular diversity and function.
- Many plant tissues are resistant to enzymatic digestion, hindering protoplast isolation for scRNA-seq.
- Field-grown crops pose challenges due to limited access to immediate laboratory facilities for protoplast preparation.
Purpose of the Study:
- To develop novel methods for preparing high-quality protoplasts from difficult-to-digest and cryopreserved plant samples for scRNA-seq.
- To overcome limitations in current plant single-cell genomics workflows.
- To expand the scope of plant single-cell multi-omics research.
Main Methods:
- Development of FX-Cell, FXcryo-Cell, and cryoFX-Cell protocols.
- Optimization of fixation buffer and minimization of RNA degradation.
- High-temperature enzymatic digestion for efficient cell wall removal.
- Cryopreservation of protoplasts for long-term storage at -80°C.
Main Results:
- Successful generation of high-quality single-cell RNA sequencing data from challenging plant tissues (rice tiller nodes, wild rice rhizomes, maize crown roots).
- Preservation of high-quality single cells after long-term cryopreservation.
- Accurate reconstruction of plant acute wounding responses at single-cell resolution.
- Circumvention of nuclei isolation, focusing on translatable messenger RNAs.
Conclusions:
- FX-Cell and its derivatives significantly expand the applicability of plant single-cell genomics.
- These methods enable scRNA-seq from a wider range of plant species and tissues, including field-grown crops.
- The developed protocols pave the way for the creation of comprehensive Plant Cell Atlases.
