Plant Nuclei Isolation for Single-Nucleus RNA Sequencing.

Area of Science:

• Plant biology
• Molecular biology
• Genomics

Background:

• Transcriptome profiling is crucial for understanding biological processes.
• Cellular heterogeneity within tissues complicates traditional transcriptome analysis.
• Single-cell RNA sequencing (scRNA-seq) has advanced transcriptome studies but has limitations in plant research.

Purpose of the Study:

• To address the limitations of scRNA-seq in plants, such as the inability to isolate protoplasts from certain cell types and the induction of wounding responses.
• To present a robust protocol for single-nucleus RNA sequencing (snRNA-seq) applicable to plant tissues.
• To facilitate broader application of high-throughput sequencing in plant research.

Main Methods:

• Isolation of nuclei from various plant tissues.
• Preparation of nuclei for library construction.
• Adaptation of snRNA-seq protocols for plant samples.
• High-throughput sequencing of isolated nuclei.

Main Results:

• snRNA-seq can be applied to a wider range of plant tissue types and species compared to scRNA-seq.
• The protocol yields nuclei suitable for subsequent library preparation and sequencing.
• While fewer transcripts are obtained per nucleus than per protoplast, snRNA-seq overcomes protoplast isolation challenges.

Conclusions:

• snRNA-seq is a valuable alternative to scRNA-seq for plant transcriptome profiling, especially for recalcitrant cell types.
• The described protocol provides a general method for preparing plant nuclei for snRNA-seq.
• This technique expands the scope of transcriptomic studies in diverse plant systems.