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

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 developmen...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Daniel Conde, Paolo M Triozzi, Kelly M Balmant, Andria L Doty, Mariza Miranda, Anthony Boullosa, Henry W Schmidt, Wendell J Pereira, Christopher Dervinis, Matias Kirst
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/fc0134b9fc2b4e0a927ddbd38aa70b45
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:fc0134b9fc2b4e0a927ddbd38aa70b45
record_format	dspace
spelling	oai:doaj.org-article:fc0134b9fc2b4e0a927ddbd38aa70b452021-12-02T20:11:21ZA robust method of nuclei isolation for single-cell RNA sequencing of solid tissues from the plant genus Populus.1932-620310.1371/journal.pone.0251149https://doaj.org/article/fc0134b9fc2b4e0a927ddbd38aa70b452021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0251149https://doaj.org/toc/1932-6203Single-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.Daniel CondePaolo M TriozziKelly M BalmantAndria L DotyMariza MirandaAnthony BoullosaHenry W SchmidtWendell J PereiraChristopher DervinisMatias KirstPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 5, p e0251149 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Daniel Conde Paolo M Triozzi Kelly M Balmant Andria L Doty Mariza Miranda Anthony Boullosa Henry W Schmidt Wendell J Pereira Christopher Dervinis Matias Kirst A robust method of nuclei isolation for single-cell RNA sequencing of solid tissues from the plant genus Populus.
description	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.
format	article
author	Daniel Conde Paolo M Triozzi Kelly M Balmant Andria L Doty Mariza Miranda Anthony Boullosa Henry W Schmidt Wendell J Pereira Christopher Dervinis Matias Kirst
author_facet	Daniel Conde Paolo M Triozzi Kelly M Balmant Andria L Doty Mariza Miranda Anthony Boullosa Henry W Schmidt Wendell J Pereira Christopher Dervinis Matias Kirst
author_sort	Daniel Conde
title	A robust method of nuclei isolation for single-cell RNA sequencing of solid tissues from the plant genus Populus.
title_short	A robust method of nuclei isolation for single-cell RNA sequencing of solid tissues from the plant genus Populus.
title_full	A robust method of nuclei isolation for single-cell RNA sequencing of solid tissues from the plant genus Populus.
title_fullStr	A robust method of nuclei isolation for single-cell RNA sequencing of solid tissues from the plant genus Populus.
title_full_unstemmed	A robust method of nuclei isolation for single-cell RNA sequencing of solid tissues from the plant genus Populus.
title_sort	robust method of nuclei isolation for single-cell rna sequencing of solid tissues from the plant genus populus.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/fc0134b9fc2b4e0a927ddbd38aa70b45
work_keys_str_mv	AT danielconde arobustmethodofnucleiisolationforsinglecellrnasequencingofsolidtissuesfromtheplantgenuspopulus AT paolomtriozzi arobustmethodofnucleiisolationforsinglecellrnasequencingofsolidtissuesfromtheplantgenuspopulus AT kellymbalmant arobustmethodofnucleiisolationforsinglecellrnasequencingofsolidtissuesfromtheplantgenuspopulus AT andrialdoty arobustmethodofnucleiisolationforsinglecellrnasequencingofsolidtissuesfromtheplantgenuspopulus AT marizamiranda arobustmethodofnucleiisolationforsinglecellrnasequencingofsolidtissuesfromtheplantgenuspopulus AT anthonyboullosa arobustmethodofnucleiisolationforsinglecellrnasequencingofsolidtissuesfromtheplantgenuspopulus AT henrywschmidt arobustmethodofnucleiisolationforsinglecellrnasequencingofsolidtissuesfromtheplantgenuspopulus AT wendelljpereira arobustmethodofnucleiisolationforsinglecellrnasequencingofsolidtissuesfromtheplantgenuspopulus AT christopherdervinis arobustmethodofnucleiisolationforsinglecellrnasequencingofsolidtissuesfromtheplantgenuspopulus AT matiaskirst arobustmethodofnucleiisolationforsinglecellrnasequencingofsolidtissuesfromtheplantgenuspopulus AT danielconde robustmethodofnucleiisolationforsinglecellrnasequencingofsolidtissuesfromtheplantgenuspopulus AT paolomtriozzi robustmethodofnucleiisolationforsinglecellrnasequencingofsolidtissuesfromtheplantgenuspopulus AT kellymbalmant robustmethodofnucleiisolationforsinglecellrnasequencingofsolidtissuesfromtheplantgenuspopulus AT andrialdoty robustmethodofnucleiisolationforsinglecellrnasequencingofsolidtissuesfromtheplantgenuspopulus AT marizamiranda robustmethodofnucleiisolationforsinglecellrnasequencingofsolidtissuesfromtheplantgenuspopulus AT anthonyboullosa robustmethodofnucleiisolationforsinglecellrnasequencingofsolidtissuesfromtheplantgenuspopulus AT henrywschmidt robustmethodofnucleiisolationforsinglecellrnasequencingofsolidtissuesfromtheplantgenuspopulus AT wendelljpereira robustmethodofnucleiisolationforsinglecellrnasequencingofsolidtissuesfromtheplantgenuspopulus AT christopherdervinis robustmethodofnucleiisolationforsinglecellrnasequencingofsolidtissuesfromtheplantgenuspopulus AT matiaskirst robustmethodofnucleiisolationforsinglecellrnasequencingofsolidtissuesfromtheplantgenuspopulus
_version_	1718374921445310464

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

Ejemplares similares