skip to main content

Title: Isolation of Plant Root Nuclei for Single Cell RNA Sequencing

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 isolatedArabidopsis thalianaroot 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 forMedicago truncatulaandSorghum bicolor

Alternate Protocol 2: Estimation of nuclei density using sNucRNA‐seq technology

more » « less
Author(s) / Creator(s):
 ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Current Protocols in Plant Biology
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    Histone post‐translational modifications (PTMs) play important roles in many biological processes, including gene regulation and chromatin dynamics, and are thus of high interest across many fields of biological research. Chromatin immunoprecipitation coupled with sequencing (ChIP‐seq) is a powerful tool to profile histone PTMsin vivo. This method, however, is largely dependent on the specificity and availability of suitable commercial antibodies. While mass spectrometry (MS)–based proteomic approaches to quantitatively measure histone PTMs have been developed in mammals and several other model organisms, such methods are currently not readily available in plants. One major challenge for the implementation of such methods in plants has been the difficulty in isolating sufficient amounts of pure, high‐quality histones, a step rendered difficult by the presence of the cell wall. Here, we developed a high‐yielding histone extraction and purification method optimized forArabidopsis thalianathat can be used to obtain high‐quality histones for MS. In contrast to other methods used in plants, this approach is relatively simple, and does not require membranes or additional specialized steps, such as gel excision or chromatography, to extract highly purified histones. We also describe methods for producing MS‐ready histone peptides through chemical labeling and digestion. Finally, we describe an optimized method to quantify and analyze the resulting histone PTM data using a modified version of EpiProfile 2.0 for Arabidopsis. In all, the workflow described here can be used to measure changes to histone PTMs resulting from various treatments, stresses, and time courses, as well as in different mutant lines. © 2022 Wiley Periodicals LLC.

    Basic Protocol 1: Nuclear isolation and histone acid extraction

    Basic Protocol 2: Peptide labeling, digestion, and desalting

    Basic Protocol 3: Histone HPLC‐MS/MS and data analysis

    more » « less
  2. Abstract

    Until recently, precise genome editing has been limited to a few organisms. The ability of Cas9 to generate double stranded DNA breaks at specific genomic sites has greatly expanded molecular toolkits in many organisms and cell types. Before CRISPR‐Cas9 mediated genome editing,P. patenswas unique among plants in its ability to integrate DNA via homologous recombination. However, selection for homologous recombination events was required to obtain edited plants, limiting the types of editing that were possible. Now with CRISPR‐Cas9, molecular manipulations inP. patenshave greatly expanded. This protocol describes a method to generate a variety of different genome edits. The protocol describes a streamlined method to generate the Cas9/sgRNA expression constructs, design homology templates, transform, and quickly genotype plants. © 2023 Wiley Periodicals LLC.

    Basic Protocol 1: Constructing the Cas9/sgRNA transient expression vector

    Alternate Protocol 1: Shortcut to generating single and pooled Cas9/sgRNA expression vectors

    Basic Protocol 2: Designing the oligonucleotide‐based homology‐directed repair (HDR) template

    Alternate Protocol 2: Designing the plasmid‐based HDR template

    Basic Protocol 3: Inducing genome editing by transforming CRISPR vector intoP. patensprotoplasts

    Basic Protocol 4: Identifying edited plants.

    more » « less
  3. Abstract Background

    The genetic information contained in the genome of an organism is organized in genes and regulatory elements that control gene expression. The genomes of multiple plants species have already been sequenced and the gene repertory have been annotated, however,cis-regulatory elements remain less characterized, limiting our understanding of genome functionality. These elements act as open platforms for recruiting both positive- and negative-acting transcription factors, and as such, chromatin accessibility is an important signature for their identification.


    In this work we developed a transgenic INTACT [isolation of nuclei tagged in specific cell types] system in tetraploid wheat for nuclei purifications. Then, we combined the INTACT system together with the assay for transposase-accessible chromatin with sequencing [ATAC-seq] to identify open chromatin regions in wheat root tip samples. Our ATAC-seq results showed a large enrichment of open chromatin regions in intergenic and promoter regions, which is expected for regulatory elements and that is similar to ATAC-seq results obtained in other plant species. In addition, root ATAC-seq peaks showed a significant overlap with a previously published ATAC-seq data from wheat leaf protoplast, indicating a high reproducibility between the two experiments and a large overlap between open chromatin regions in root and leaf tissues. Importantly, we observed overlap between ATAC-seq peaks andcis-regulatory elements that have been functionally validated in wheat, and a good correlation between normalized accessibility and gene expression levels.


    We have developed and validated an INTACT system in tetraploid wheat that allows rapid and high-quality nuclei purification from root tips. Those nuclei were successfully used to performed ATAC-seq experiments that revealed open chromatin regions in the wheat genome that will be useful to identify cis-regulatory elements. The INTACT system presented here will facilitate the development of ATAC-seq datasets in other tissues, growth stages, and under different growing conditions to generate a more complete landscape of the accessible DNA regions in the wheat genome.

    more » « less
  4. Abstract

    Class II major histocompatibility complex peptide (MHC‐IIp) multimers are precisely engineered reagents used to detect T cells specific for antigens from pathogens, tumors, and self‐proteins. While the related Class I MHC/peptide (MHC‐Ip) multimers are usually produced from subunits expressed inE. coli, most Class II MHC alleles cannot be produced in bacteria, and this has contributed to the perception that MHC‐IIp reagents are harder to produce. Herein, we present a robust constitutive expression system for soluble biotinylated MHC‐IIp proteins that uses stable lentiviral vector−transduced derivatives of HEK‐293T cells. The expression design includes allele‐specific peptide ligands tethered to the amino‐terminus of the MHC‐II β chain via a protease‐cleavable linker. Following cleavage of the linker, HLA‐DM is used to catalyze efficient peptide exchange, enabling high‐throughput production of many distinct MHC‐IIp complexes from a single production cell line. Peptide exchange is monitored using either of two label‐free methods, native isoelectric focusing gel electrophoresis or matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry of eluted peptides. Together, these methods produce MHC‐IIp complexes that are highly homogeneous and that form the basis for excellent MHC‐IIp multimer reagents. © 2021 Wiley Periodicals LLC.

    This article was corrected on 19 July 2022. See the end of the full text for details.

    Basic Protocol 1: Lentivirus production and expression line creation

    Support Protocol 1: Six‐well assay for estimation of production cell line yield

    Support Protocol 2: Universal ELISA for quantifying proteins with fused leucine zippers and His‐tags

    Basic Protocol 2: Cultures for production of Class II MHC proteins

    Basic Protocol 3: Purification of Class II MHC proteins by anti‐leucine zipper affinity chromatography

    Alternate Protocol 1: IMAC purification of His‐tagged Class II MHC

    Support Protocol 3: Protein concentration measurements and adjustments

    Support Protocol 4: Polishing purification by anion‐exchange chromatography

    Support Protocol 5: Estimating biotinylation percentage by streptavidin precipitation

    Basic Protocol 4: Peptide exchange

    Basic Protocol 5: Analysis of peptide exchange by matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry

    Alternate Protocol 2: Native isoelectric focusing to validate MHC‐II peptide loading

    Basic Protocol 6: Multimerization

    Basic Protocol 7: Staining cells with Class II MHC tetramers

    more » « less
  5. Abstract

    Extracellular vesicles (EVs) in plants have emerged as key players in cell‐to‐cell communication and cross‐kingdom RNAi between plants and pathogens by facilitating the exchange of RNA, proteins, and other molecules. In addition to their role in intercellular communication, plant EVs also show promise as potential therapeutics and indicators of plant health. However, plant EVs exhibit significant heterogeneity in their protein markers, size, and biogenesis pathways, strongly influencing their composition and functionality. While mammalian EVs can be generally classified as exosomes that are derived from multivesicular bodies (MVBs), microvesicles that are shed from the plasma membrane, or as apoptotic bodies that originate from cells undergoing apoptosis, plant EVs remain poorly studied in comparison. At least three subclasses of EVs have been identified inArabidopsisleaves to date, including Tetraspanin‐positive exosomes derived from MVBs, Penetration 1 (PEN1)‐positive EVs, and EVs derived from exocyst‐positive organelles (EXPO). Differences in the plant starting material and isolation techniques have resulted in different purities, quality, and compositions of the resulting EVs, complicating efforts to better understand the role of these EVs in plants. We performed a comparative analysis on commonly used plant EV isolation methods and have identified an effective protocol for extracting clean apoplastic washing fluid (AWF) and isolating high‐quality intact and pure EVs ofArabidopsis thaliana. These EVs can then be used for various applications or studied to assess their cargos and functionality in plants. Furthermore, this process can be easily adapted to other plant species of interest. © 2022 Wiley Periodicals LLC.

    Basic Protocol 1: Isolation of EVs from the apoplastic fluid ofArabidopsis thaliana

    Basic Protocol 2: Density gradient fractionation of EVs

    Basic Protocol 3: Immuno‐isolation of EVs usingArabidopsistetraspanin 8 (TET8) antibody

    more » « less