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Development of High-Quality Nuclei Isolation to Study Plant Root–Microbe Interaction for Single-Nuclei Transcriptomic Sequencing in Soybean

Single-nucleus RNA sequencing (sNucRNA-seq) is an emerging technology that has been rapidly adopted and demonstrated to be a powerful tool for detailed characterization of each cell- and sub cell-types in complex tissues of higher eukaryotes. sNucRNA-seq has also been used to dissect cell-type-speci...

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Autores principales: D’Agostino, Leonidas W., Yong-Villalobos, Lenin, Herrera-Estrella, Luis, Patil, Gunvant B.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10346333/
https://www.ncbi.nlm.nih.gov/pubmed/37447027
http://dx.doi.org/10.3390/plants12132466
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author D’Agostino, Leonidas W.
Yong-Villalobos, Lenin
Herrera-Estrella, Luis
Patil, Gunvant B.
author_facet D’Agostino, Leonidas W.
Yong-Villalobos, Lenin
Herrera-Estrella, Luis
Patil, Gunvant B.
author_sort D’Agostino, Leonidas W.
collection PubMed
description Single-nucleus RNA sequencing (sNucRNA-seq) is an emerging technology that has been rapidly adopted and demonstrated to be a powerful tool for detailed characterization of each cell- and sub cell-types in complex tissues of higher eukaryotes. sNucRNA-seq has also been used to dissect cell-type-specific transcriptional responses to environmental or developmental signals. In plants, this technology is being utilized to identify cell-type-specific trajectories for the study of several tissue types and important traits, including the single-cell dissection of the genetic determinants regulating plant–microbe interactions. The isolation of high-quality nuclei is one of the prerequisite steps to obtain high-quality sNucRNA-seq results. Although nuclei isolation from several plant tissues is well established, this process is highly troublesome when plant tissues are associated with beneficial or pathogenic microbes. For example, root tissues colonized with rhizobium bacteria (nodules), leaf tissue infected with bacterial or fungal pathogens, or roots infected with nematodes pose critical challenges to the isolation of high-quality nuclei and use for downstream application. Therefore, isolation of microbe-free, high-quality nuclei from plant tissues are necessary to avoid clogging or interference with the microfluidic channel (e.g., 10× Genomics) or particle-templated emulsion that are used in sNucRNA-seq platforms. Here, we developed a simple, effective, and efficient method to isolate high-quality nuclei from soybean roots and root nodules, followed by washing out bacterial contamination. This protocol has been designed to be easily implemented into any lab environment, and it can also be scaled up for use with multiple samples and applicable to a variety of samples with the presence of microbes. We validated this protocol by successfully generating a barcoded library using the 10× Genomics microfluidic platform from tissue subjected to this procedure. This workflow was developed to provide an accessible alternative to instrument-based approaches (e.g., fluorescent cell sorting) and will expand the ability of researchers to perform experiments such as sNucRNA-seq and sNucATAC-seq on inherently heterogeneous plant tissue samples.
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spelling pubmed-103463332023-07-15 Development of High-Quality Nuclei Isolation to Study Plant Root–Microbe Interaction for Single-Nuclei Transcriptomic Sequencing in Soybean D’Agostino, Leonidas W. Yong-Villalobos, Lenin Herrera-Estrella, Luis Patil, Gunvant B. Plants (Basel) Protocol Single-nucleus RNA sequencing (sNucRNA-seq) is an emerging technology that has been rapidly adopted and demonstrated to be a powerful tool for detailed characterization of each cell- and sub cell-types in complex tissues of higher eukaryotes. sNucRNA-seq has also been used to dissect cell-type-specific transcriptional responses to environmental or developmental signals. In plants, this technology is being utilized to identify cell-type-specific trajectories for the study of several tissue types and important traits, including the single-cell dissection of the genetic determinants regulating plant–microbe interactions. The isolation of high-quality nuclei is one of the prerequisite steps to obtain high-quality sNucRNA-seq results. Although nuclei isolation from several plant tissues is well established, this process is highly troublesome when plant tissues are associated with beneficial or pathogenic microbes. For example, root tissues colonized with rhizobium bacteria (nodules), leaf tissue infected with bacterial or fungal pathogens, or roots infected with nematodes pose critical challenges to the isolation of high-quality nuclei and use for downstream application. Therefore, isolation of microbe-free, high-quality nuclei from plant tissues are necessary to avoid clogging or interference with the microfluidic channel (e.g., 10× Genomics) or particle-templated emulsion that are used in sNucRNA-seq platforms. Here, we developed a simple, effective, and efficient method to isolate high-quality nuclei from soybean roots and root nodules, followed by washing out bacterial contamination. This protocol has been designed to be easily implemented into any lab environment, and it can also be scaled up for use with multiple samples and applicable to a variety of samples with the presence of microbes. We validated this protocol by successfully generating a barcoded library using the 10× Genomics microfluidic platform from tissue subjected to this procedure. This workflow was developed to provide an accessible alternative to instrument-based approaches (e.g., fluorescent cell sorting) and will expand the ability of researchers to perform experiments such as sNucRNA-seq and sNucATAC-seq on inherently heterogeneous plant tissue samples. MDPI 2023-06-28 /pmc/articles/PMC10346333/ /pubmed/37447027 http://dx.doi.org/10.3390/plants12132466 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Protocol
D’Agostino, Leonidas W.
Yong-Villalobos, Lenin
Herrera-Estrella, Luis
Patil, Gunvant B.
Development of High-Quality Nuclei Isolation to Study Plant Root–Microbe Interaction for Single-Nuclei Transcriptomic Sequencing in Soybean
title Development of High-Quality Nuclei Isolation to Study Plant Root–Microbe Interaction for Single-Nuclei Transcriptomic Sequencing in Soybean
title_full Development of High-Quality Nuclei Isolation to Study Plant Root–Microbe Interaction for Single-Nuclei Transcriptomic Sequencing in Soybean
title_fullStr Development of High-Quality Nuclei Isolation to Study Plant Root–Microbe Interaction for Single-Nuclei Transcriptomic Sequencing in Soybean
title_full_unstemmed Development of High-Quality Nuclei Isolation to Study Plant Root–Microbe Interaction for Single-Nuclei Transcriptomic Sequencing in Soybean
title_short Development of High-Quality Nuclei Isolation to Study Plant Root–Microbe Interaction for Single-Nuclei Transcriptomic Sequencing in Soybean
title_sort development of high-quality nuclei isolation to study plant root–microbe interaction for single-nuclei transcriptomic sequencing in soybean
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10346333/
https://www.ncbi.nlm.nih.gov/pubmed/37447027
http://dx.doi.org/10.3390/plants12132466
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