Isolation of Plant Root Nuclei for Single Cell RNA Sequencing

Abstract

The characterization of the transcriptional similarities and differences existing between plant cells and cell types is important to better understand the biology of each cell composing the plant, to reveal new molecular mechanisms controlling gene activity, and to ultimately implement meaningful strategies to enhance plant cell biology. To gain a deeper understanding of the regulation of plant gene activity, the individual transcriptome of each plant cell needs to be established. Until recently, single cell approaches were mostly limited to bulk transcriptomic studies on selected cell types. Accessing specific cell types required the development of labor‐intensive strategies. Recently, single cell sequencing strategies were successfully applied on isolated Arabidopsis thaliana root protoplasts. However, this strategy relies on the successful isolation of viable protoplasts upon the optimization of the enzymatic cocktails required to digest the cell wall and on the compatibility of fragile plant protoplasts with the use of microfluidic systems to generate single cell transcriptomic libraries. To overcome these difficulties, we present a simple and fast alternative strategy: the isolation and use of plant nuclei to access meaningful transcriptomic information from plant cells. This protocol was specifically developed to enable the use of the plant nuclei with 10× Genomics’ Chromium technology partitions technology. Briefly, the plant nuclei are released from the root by chopping into a nuclei isolation buffer before purification by filtration then nuclei sorting. Upon sorting, the nuclei are resuspended in a low divalent ion buffer compatible with the Chromium technology in order to create single nuclei ribonucleic acid‐sequencing libraries (sNucRNA‐seq). © 2020 Wiley Periodicals LLC. Basic Protocol 1: Arabidopsis seed sterilization and planting Basic Protocol 2: Nuclei isolation from Arabidopsis roots Basic Protocol 3: Fluorescent‐activated nuclei sorting (FANS) purification Support Protocol: Estimation of nuclei density using Countess II automated cell counter Alternate Protocol 1: Proper growth conditions for Medicago truncatula and Sorghum bicolor Alternate Protocol 2: Estimation of nuclei density using sNucRNA‐seq technology

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

“…In plants, research that use isolated nuclei for microfluidics snRNA-seq methods remain very limited. Using the 10× Genomics Chromium system, an average of 1,126 expressed genes per nucleus were obtained using Arabidopsis roots [20,22].…”

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

“…Using the 10× Genomics Chromium system, an average of 1,126 expressed genes per nucleus were obtained using Arabidopsis roots [20,22]. So far, most scRNA-seq analyses performed in plants used protoplasts from Arabidopsis plant tissues [9][10][11][12].…”

“…After samples were filtered through miracloth and a 40 μ m strainer, they were washed to remove ambient RNA from cellular cytoplasm or that may have leaked from nuclei during the isolation process. Standard protocols for plant nuclei isolation include centrifugation steps that range between 500-1500 g, for 5-10 min [18,20,22,23]. In the nuclei washes we centrifuged samples at 600 g for 5 min, to minimize damage to nuclei while pelleting them at the bottom of the tubes.…”

Single-nucleus full-length RNA profiling in plants incorporates isoform information to facilitate cell type identification

“…As a proof-of-concept demonstration in plant, our results showed that protoplasting-free large-scale single-nucleus sequencing is sufficient for cell type classification and marker gene identification in Arabidopsis roots. As we are preparing this manuscript, several groups have also recently adopted the nuclei-based protoplasting-free strategy independently to investigate various plant tissues[35-39]. Eliminating protoplasting as a prerequisite would enable large-scale single-cell profiling on a wide range of tissues and plant species.…”