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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| Format: | Article |
| Language: | English |
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MDPI AG
2023-06-01
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| Series: | Plants |
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| Online Access: | https://www.mdpi.com/2223-7747/12/13/2466 |
| _version_ | 1797591021230489600 |
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| author | Leonidas W. D’Agostino Lenin Yong-Villalobos Luis Herrera-Estrella Gunvant B. Patil |
| author_facet | Leonidas W. D’Agostino Lenin Yong-Villalobos Luis Herrera-Estrella Gunvant B. Patil |
| author_sort | Leonidas W. D’Agostino |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-11T01:31:47Z |
| format | Article |
| id | doaj.art-e382758109a34b4ba7b907e0736d6a49 |
| institution | Directory Open Access Journal |
| issn | 2223-7747 |
| language | English |
| last_indexed | 2024-03-11T01:31:47Z |
| publishDate | 2023-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Plants |
| spelling | doaj.art-e382758109a34b4ba7b907e0736d6a492023-11-18T17:17:52ZengMDPI AGPlants2223-77472023-06-011213246610.3390/plants12132466Development of High-Quality Nuclei Isolation to Study Plant Root–Microbe Interaction for Single-Nuclei Transcriptomic Sequencing in SoybeanLeonidas W. D’Agostino0Lenin Yong-Villalobos1Luis Herrera-Estrella2Gunvant B. Patil3Institute of Genomics for Crop Abiotic Stress Tolerance, Department of Plant and Soil Science, Texas Tech University, Lubbock, TX 79409, USAInstitute of Genomics for Crop Abiotic Stress Tolerance, Department of Plant and Soil Science, Texas Tech University, Lubbock, TX 79409, USAInstitute of Genomics for Crop Abiotic Stress Tolerance, Department of Plant and Soil Science, Texas Tech University, Lubbock, TX 79409, USAInstitute of Genomics for Crop Abiotic Stress Tolerance, Department of Plant and Soil Science, Texas Tech University, Lubbock, TX 79409, USASingle-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.https://www.mdpi.com/2223-7747/12/13/2466soybeannodulesingle cellsNucRNAnuclei10× |
| spellingShingle | Leonidas W. D’Agostino Lenin Yong-Villalobos Luis Herrera-Estrella Gunvant B. Patil Development of High-Quality Nuclei Isolation to Study Plant Root–Microbe Interaction for Single-Nuclei Transcriptomic Sequencing in Soybean Plants soybean nodule single cell sNucRNA nuclei 10× |
| 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 | soybean nodule single cell sNucRNA nuclei 10× |
| url | https://www.mdpi.com/2223-7747/12/13/2466 |
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