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

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.