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Sci Rep
2021 Jan 18;111:1681. doi: 10.1038/s41598-021-81238-z.
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An optimised method for intact nuclei isolation from diatoms.
Annunziata R
,
Balestra C
,
Marotta P
,
Ruggiero A
,
Manfellotto F
,
Benvenuto G
,
Biffali E
,
Ferrante MI
.
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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.
Figure 1. Isolation of intact nuclei from Pseudo-nitzschia multistriata, Phaeodactylum tricornutum, and Chaetoceros diadema. Bright-field and fluorescent microscopy images of intact cells, sonicated cells and sorted nuclei for P. multistriata (a), P. tricornutum (b) and C. diadema (c), scale bar 5 μm. Dot plots (up) and histograms (down) of flow cytometry analysis for nuclei isolation in P. multistriata (d), P. tricornutum (e) and C. diadema (f). In dark blue P1 indicates the gate containing extracted nuclei; in light blue P2 indicates the gate containing cellular residues. White and black arrows point to extracted nuclei. BF (Bright Field), SG (SYBR Green I), SSC (Side-Scatter). Graphs in (d), (e) and (f) were drawn with FCS Express 6 Flow v 6.06.0025, DeNovo Software, USA.
Figure 2. Microscopy analysis of P. multistriata cell and isolated nuclei. Confocal (a–f) and SEM (g–i) images of P. multistriata isolated cell (a–c) and nuclei (d–i) after the FAC-sorting procedure. In C chlorophyll autofluorescence is visible in red. BF (bright field), SG (SYBR Green I).
Figure 3. Confocal microscopy analysis of C. diadema cells and isolated nuclei. Bright-field and fluorescence microscopy images of intact cell chains (a–c), single cell (d–f) and sorted nuclei (g–i) for C. diadema. In (c) and (f) chlorophyll autofluorescence is visible in red. BF (Bright Field), SG (SYBR Green I).
Figure 4. Quality control of genomic DNA extracted from P. multistriata, P. tricornutum, and C. diadema. High sensitivity DNA electrophoresis (Agilent 2100 Bioanalyzer System) of gDNAs extracted from intact cells and isolated nuclei from P. multistriata, P. tricornutum and C. diadema. N (nuclei), C (cells), bp (base pairs). In purple and green the higher and the lower bands of the DNA-ladder used as reference.
Figure 5. Intact nuclei isolation from diatom cells: the experimental workflow. Step by step protocol and useful warnings and tips to isolate intact nuclei from diatom cells. Main steps are indicated on the left: diatoms cells are harvested via centrifugation; NH4F treatment is performed to weaken diatom cell wall; sonication is applied to destroy frustules allowing nuclei extraction which is verified using epifluorescence microscopy; nuclei are sorted by FACS from a mixed sample containing also cellular debris and, in some cases, bacteria, obtaining a clean preparation of isolated nuclei. ASW (Artificial Sea Water), RT (Room Temperature), SG (SYBR Green I), NIB (Nuclei Isolation Buffer), FACS (Fluorescence Activated Cell Sorting), SSC (Side-Scatter). Images used for the figure were adapted from “Library of Science and Medical Illustrations CC BY-NC-SA 4.0”.
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