Plant Nuclei Isolation for Single-Nucleus RNA Sequencing.

Transcriptome profiling has been significantly hampered by the heterogeneity among individual cells within a tissue or an organ. Recent advances in single cell transcriptome profiling have significantly advanced our understanding of the transcriptome. However, plant single-cell RNA sequencing (scRNA-seq) relies on the isolation of protoplasts, which is not only impossible for many cell types but also induces acute wounding responses. To solve these problems, single-nucleus RNA sequencing (snRNA-seq) has been applied to plant research, in which nuclei are isolated and subject to encapsulation and profiling. Compared with scRNA-seq, snRNA-seq can be applied to a wider range of tissue types and plant species. Nevertheless, fewer transcripts can be obtained from each nucleus than each protoplast. In this chapter, we describe a detailed and general protocol to prepare nuclei from plant tissues that are ready for subsequent library construction and high-throughput sequencing.