A robust method of nuclei isolation for single-cell RNA sequencing of solid tissues from the plant genus Populus

Abstract

Single-cell transcriptome analysis has been extensively applied in humans and animal models to uncover gene expression heterogeneity between the different cell types of a tissue or an organ. It demonstrated its capability to discover key regulatory elements that determine cell fate during developmental programs. Single-cell analysis requires the isolation and labeling of the messenger RNA (mRNA) derived from each cell. These challenges were primarily addressed in mammals by developing microfluidic-based approaches. For plant species whose cells contain cell walls, these approaches have generally required the generation of isolated protoplasts. Many plant tissues’ secondary cell wall hinders enzymatic digestion required for individual protoplast isolation, resulting in an unequal representation of cell types in a protoplast population. This limitation is especially critical for cell types located in the inner layers of a tissue or the inner tissues of an organ. Consequently, single-cell RNA sequencing (scRNA-seq) studies using microfluidic approaches in plants have mainly been restricted to Arabidopsis roots, for which well-established procedures of protoplast isolation are available. Here we present a simple alternative approach to generating high-quality protoplasts from plant tissue by characterizing the mRNA extracted from individual nuclei instead of whole cells. We developed the protocol using two different plant materials with varying cellular complexity levels and cell wall structure, Populus shoot apices, and more lignified stems. Using the 10× Genomics Chromium technology, we show that this procedure results in intact mRNA isolation and limited leakage, with a broad representation of individual cell transcriptomes.

Single-cell analysis resolves the genetic program of primary vascular system formation in hybrid poplar

“…We show that the isolation and analysis of individual nuclei result in the detection of diverse cell types and numbers of transcripts to enable their discrimination at a resolution comparable with whole cells in plants. Moreover, we demonstrated that this procedure can be applied to lignified Populus stems (13). The reported transcriptomes of shoot apex procambial cells, in combination with the feasibility of our nuclei isolation protocol to explore the single-cell transcriptomic in the stem, constitute an unpreceded opportunity to dissect the missing link between primary and secondary growth, including how cambium develops from procambium at the initiation of secondary growth.…”

“…We harvested 20 shoot apices from 3-weeks-old in vitro grown hybrid poplar (Populus tremula × alba) plants and performed high-throughput, microfluidic-based single-nuclei RNA sequencing (snRNA-seq) using the 10× Genomics Chromium technology as we described previously (13). For all quality control and analytical steps, we used Asc-Seurat (14), a comprehensive web application that encapsulates a series of tools for scRNA-seq data analysis.…”

“…To perform nuclei isolation, we followed the previously developed protocol for poplar shoot apices (13).…”

“…Twenty thousand nuclei were loaded to the 10X Genomics Chromium to generate the snRNA-seq library, following the 10× Genomics Chromium Single Cell v3.1 protocol. The cDNA reaction was prepared as described previously (13).…”

“…Here we applied a novel approach to isolate nuclei from complex plant tissues (13), to dissect the transcriptome profile of the hybrid poplar (Populus tremula × alba) vegetative shoot apex at single-cell resolution. We inferred the developmental trajectories that occur during the generation of new tissues in the SAM.…”