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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 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.

SUBMITTER: Annunziata R

PROVIDER: S-EPMC7813820 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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An optimised method for intact nuclei isolation from diatoms.

Annunziata Rossella R Balestra Cecilia C Marotta Pina P Ruggiero Antonella A Manfellotto Francesco F Benvenuto Giovanna G Biffali Elio E Ferrante Maria Immacolata MI

Scientific reports 20210118 1

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 prese ...[more]

PMID: 33462289

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Project description:Recent studies have highlighted the extraordinary cell type diversity that exists within mammalian organs, yet the molecular drivers of such heterogeneity remain elusive. To address this issue, much attention has been focused on profiling the transcriptome and epigenome of individual cell types. However, standard cell type isolation methods based on surface or fluorescent markers remain problematic for cells residing within organs with significant connective tissue. Since the nucleus contains both genomic and transcriptomic information, the isolation of nuclei tagged in specific cell types (INTACT) method provides an attractive solution. Although INTACT has been successfully applied to plants, flies, zebrafish, frogs, and mouse brain and adipose tissue, broad use across mammalian organs remains challenging. Here we describe the PAN-INTACT method, which can be used to isolate cell type specific nuclei from fibrous mouse organs, which are particularly problematic. As a proof-of-concept, we demonstrate successful isolation of cell type-specific nuclei from the mouse heart, which contains substantial connective tissue and harbors multiple cell types, including cardiomyocytes, fibroblasts, endothelial cells, and epicardial cells. Compared to established techniques, PAN-INTACT allows more rapid isolation of cardiac nuclei to facilitate downstream applications. We show cell type-specific isolation of nuclei from the hearts of Nkx2-5Cre/+; R26Sun1-2xsf-GFP-6xmyc/+ mice, which we confirm by expression of lineage markers. Furthermore, we perform Assay for Transposase Accessible Chromatin (ATAC)-Seq to provide high-fidelity chromatin accessibility maps of Nkx2-5+ nuclei. To extend the applicability of PAN-INTACT, we also demonstrate successful isolation of Wt1+ podocytes from adult kidney. Taken together, our data suggest that PAN-INTACT is broadly applicable for profiling the transcriptional and epigenetic landscape of specific cell types. Thus, we envision that our method can be used to systematically probe mechanistic details of cell type-specific functions within individual organs of intact mice.

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An optimised method for intact nuclei isolation from diatoms.

Publications

An optimised method for intact nuclei isolation from diatoms.

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