Single-nucleus RNA sequencing is a recently developed method of profiling gene expression in cells that are hard to isolate, or tissue that has been archived using isolated nuclei. The method involves obtaining nuclei suspension by extracting the nuclei from tissue. The nuclei suspension is then stained with DAPI and observed under fluorescence microscope to assess the integrity and brightness of the nuclei. This allows for the evaluation of the quality of the nuclei, and the method is used to conduct transcriptome research on the nuclei that meet the quality control requirements.

Currently, Sun et al [1], Neumann et al [2], and Farmer et al [3] have published high-scoring articles using snRNA-seq. These research results have strongly demonstrated the feasibility and reliability of single-nucleus RNA sequencing in plant research. At the same time, it also demonstrate the advantages of snRNA-seq research. Compared with scRNA-seq of protoplasts, snRNA-seq has obvious advantages for plant research.

The length of time for preparation of single-cell suspension affects the experimental efficiency. Due to the presence of cell walls, plants have always relied on enzymatic digestion of cell walls to prepare protoplasts for scRNA-seq studies. However, cell wall digestion often takes 1-2 hours, and prolonged enzymatic digestion reduces the efficiency of the experiment

In contrast, single nuclei suspension preparation is simpler and faster. Single nucleus suspension preparation includes several processes: harvesting tissue, brokening tissue, preparing crude nuclei, pacificating crude nuclei, and microscopic examination. Physical fragmentation (grinding and cutting methods) is a common method for the preparation of nuclei suspensions, which can quickly release nuclei within a few minutes, which greatly shortens the preparation time of nuclei suspensions and improves the efficiency of experiments.

In addition, the nucleus has a bilayer structure and the nucleus is small in size, so the nucleus membrane is strong and not easily broken, which makes it easier to obtain high-quality nuclei suspensions.

Low requirements for a wider range of tissues

Although protoplasts are a common method for the preparation of single cell suspensions for plant, not all tissue samples can successfully obtain protoplasts. To obtain intact protoplasts with high cell viability, fresh and young samples are generally required. This is because the enzymatic preparation of protoplasts is difficult to dissociate from samples that are not fresh or young, which results in relatively low protoplast yield and cell viability. However, it is difficult to carry out single-cell studies on fresh samples obtained in a timely manner during the research process，ang the samples obtained are not always young parts, which results in many plant tissues not being available for studies on single cells. Compared to protoplasts, single nuclei preparation requires relatively less sample. For snRNA-seq studies based on single nuclei, the physical fragmentation protocol for preparing single nuclei is not only suitable for fresh samples that can be obtained in time,  but is also very friendly for frozen storage. In addition, physical fragmentation of more complex or fibrotic tissues, such as flowers, fruits, and stems, can be easily used to obtain single nuclei suspensions for snRNA-seq. Thus, the lower sample requirements allow snRNA-seq to be applied to a wider range of tissue samples.

Less bias in sample capture cell types

When conducting single-cell studies, the bias of the captured cell type can affect the accuracy of the results. The bias in cell type capture refers to the fact that some cells are easily captured and some are difficult to capture. In the process of enzymatic digestion of cell wall to prepare protoplasts, some cells are resistant to enzymes making their cell wall difficult to be digested by enzymatic digestion, while some cell subpopulations are difficult to come into direct contact with enzymatic digestion solution, so these cells are difficult to obtain protoplasts, which leads to a certain bias of cell types captured by scRNA-seq. In contrast, snRNA-seq is able to reduce the bias of captured cell types. Physical fragmentation of nuclei is a simple and brutal method that is "uniform" for all tissue types and is effective in capturing a wide range of cell types. For example, in a study by Farmer et al [3], snRNA-seq of Arabidopsis roots yielded more cell subpopulations than scRNA-seq.

Reducing anthropogenically introduced changes in gene expression

Enzymatic digestion of plant cell walls produces a stress response to the protoplasts. The environment that produces harm to the plant is called adversity, also known as stress, and the process of enzymatic digestion of the cell wall is a stress response. When cells are subjected to enzymatic digestion, their original living conditions are altered and they tend to adapt to the stress conditions by a series of stress responses through changes in gene expression, which are transcriptional deviations introduced by human factors. Therefore, the enzymatic preparation of protoplasts may bring about anthropogenic gene expression changes. However, the single nucleus transcriptome obtains the expression of genes at a certain moment, and does not change gene expression due to changes in external conditions. Therefore, snRNA-seq can reduce the gene expression changes introduced by human factors, and the obtained gene expression level is closer to the expression of the original state of the plant.

Not affected by the limitation of cell size

Currently, techniques based on microdroplet or microfluidic principles are the main single-cell research methods. Either method-based, large size cells are more difficult to capture. This is because large size cells tend to clog pore channels, while small size cells can more easily pass through the pore channels and thus capture cellular mRNA. Due to the size difference of plant cells themselves and the effect of osmotic pressure of the solution, protoplasts cells that lose their cell walls tend to become larger, and these large size protoplasts can be used for scRNA-seq through protoplasts if they produce clogged pores when they are mounted, thus reduce the cell capture rate. In contrast, snRNA-seq is much less affected by the size of the nucleus. Compared to single cells, single nuclei are smaller in size and less susceptible to change in size due to factors such as osmotic pressure of the solution, so the small size of single nuclei are more likely to pass through the pore and less likely to cause plugging. Thus, in contrast, snRNA-seq is virtually unaffected by the limitations of cell size.

Data reliable

Several studies [1-3] have shown that snRNA-seq not only yields meaningful biological data, but also converges with the analysis of data obtained from scRNA-seq, which demonstrates the reliability of the quality of data obtained from snRNA-seq.

Summary

In conclusion, compared with scRNA-seq, snRNA-seq has the advantages of simple sample preparation, high experimental efficiency, compatibility, applicability to frozen samples, reduced human-induced gene expression alterations, and lower cell type bias in plant. For tissues， such as roots and leaves where protoplasts can be easily prepared, scRNA-seq studies can be carried out preferentially. While for tissues that are difficult to prepare protoplasts such as flowers, fruits, large diameter cells, non-juvenile or severely fibrotic, snRNA-seq studies can be selected.

References

[1] Sun G, Xia M, Li J, et al. The maize single-nucleus transcriptome comprehensively describes signaling networks governing movement and development of grass stomata. Plant Cell. 2022 Apr 26;34(5):1890-1911.
[2] Neumann M, Xu X, Smaczniak C, et al. A 3D gene expression atlas of the floral meristem based on spatial reconstruction of single nucleus RNA sequencing data. Nature Communations. 2022 May 20;13(1):2838.
[3] Farmer A，Thibivilliers S，Ryu K H, et al.Single-nucleus RNA and ATAC sequencing reveals the impact of chromatin accessibility on gene expression inArabidopsis roots at the single-cell level. Mol Plant. 2021 Mar 1;14(3):372-383.