An optimised method for intact nuclei isolation from diatoms

Abstract Due to their abundance in the oceans, their extraordinary biodiversity and the increasing use for biotech applications, the study of diatom biology is receiving more and more attention in the recent years. One of the limitations in developing molecular tools for diatoms lies in the peculiar...

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Autores principales:	Rossella Annunziata, Cecilia Balestra, Pina Marotta, Antonella Ruggiero, Francesco Manfellotto, Giovanna Benvenuto, Elio Biffali, Maria Immacolata Ferrante
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/a762f558e0c947fd88848ca27c0b0b3c
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spelling	oai:doaj.org-article:a762f558e0c947fd88848ca27c0b0b3c2021-12-02T14:07:47ZAn optimised method for intact nuclei isolation from diatoms10.1038/s41598-021-81238-z2045-2322https://doaj.org/article/a762f558e0c947fd88848ca27c0b0b3c2021-01-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-81238-zhttps://doaj.org/toc/2045-2322Abstract Due to their abundance in the oceans, their extraordinary biodiversity and the increasing use for biotech applications, the study of diatom biology is receiving more and more attention in the recent years. One of the limitations in developing molecular tools for diatoms lies in the peculiar nature of their cell wall, that is made of silica and organic molecules and that hinders the application of standard methods for cell lysis required, for example, to extract organelles. In this study we present a protocol for intact nuclei isolation from diatoms that was successfully applied to three different species: two pennates, Pseudo-nitzschia multistriata and Phaeodactylum tricornutum, and one centric diatom species, Chaetoceros diadema. Intact nuclei were extracted by treatment with acidified NH4F solution combined to low intensity sonication pulses and separated from cell debris via FAC-sorting upon incubation with SYBR Green. Microscopy observations confirmed the integrity of isolated nuclei and high sensitivity DNA electrophoresis showed that genomic DNA extracted from isolated nuclei has low degree of fragmentation. This protocol has proved to be a flexible and versatile method to obtain intact nuclei preparations from different diatom species and it has the potential to speed up applications such as epigenetic explorations as well as single cell (“single nuclei”) genomics, transcriptomics and proteomics in different diatom species.Rossella AnnunziataCecilia BalestraPina MarottaAntonella RuggieroFrancesco ManfellottoGiovanna BenvenutoElio BiffaliMaria Immacolata FerranteNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Rossella Annunziata Cecilia Balestra Pina Marotta Antonella Ruggiero Francesco Manfellotto Giovanna Benvenuto Elio Biffali Maria Immacolata Ferrante An optimised method for intact nuclei isolation from diatoms
description	Abstract Due to their abundance in the oceans, their extraordinary biodiversity and the increasing use for biotech applications, the study of diatom biology is receiving more and more attention in the recent years. One of the limitations in developing molecular tools for diatoms lies in the peculiar nature of their cell wall, that is made of silica and organic molecules and that hinders the application of standard methods for cell lysis required, for example, to extract organelles. In this study we present a protocol for intact nuclei isolation from diatoms that was successfully applied to three different species: two pennates, Pseudo-nitzschia multistriata and Phaeodactylum tricornutum, and one centric diatom species, Chaetoceros diadema. Intact nuclei were extracted by treatment with acidified NH4F solution combined to low intensity sonication pulses and separated from cell debris via FAC-sorting upon incubation with SYBR Green. Microscopy observations confirmed the integrity of isolated nuclei and high sensitivity DNA electrophoresis showed that genomic DNA extracted from isolated nuclei has low degree of fragmentation. This protocol has proved to be a flexible and versatile method to obtain intact nuclei preparations from different diatom species and it has the potential to speed up applications such as epigenetic explorations as well as single cell (“single nuclei”) genomics, transcriptomics and proteomics in different diatom species.
format	article
author	Rossella Annunziata Cecilia Balestra Pina Marotta Antonella Ruggiero Francesco Manfellotto Giovanna Benvenuto Elio Biffali Maria Immacolata Ferrante
author_facet	Rossella Annunziata Cecilia Balestra Pina Marotta Antonella Ruggiero Francesco Manfellotto Giovanna Benvenuto Elio Biffali Maria Immacolata Ferrante
author_sort	Rossella Annunziata
title	An optimised method for intact nuclei isolation from diatoms
title_short	An optimised method for intact nuclei isolation from diatoms
title_full	An optimised method for intact nuclei isolation from diatoms
title_fullStr	An optimised method for intact nuclei isolation from diatoms
title_full_unstemmed	An optimised method for intact nuclei isolation from diatoms
title_sort	optimised method for intact nuclei isolation from diatoms
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/a762f558e0c947fd88848ca27c0b0b3c
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An optimised method for intact nuclei isolation from diatoms

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