FindingNemo in OneDay: Ultra-Long ONT Library Preparation from Cell to Flowcell in One Day v1

This protocol is focussed on the isolation of ultra-high molecular weight (UHMW) DNA, library preparation and clean up ready for sequencing on the Oxford Nanopore Technology (ONT) platforms, all within one working day. This protocol is optimised for human cell lines. Summary of the workflow:

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FindingNemo: A Toolkit of CoHex- and Glass Bead-based Protocols for Ultra-Long Sequencing on ONT Platforms v1

10.17504/protocols.io.bxwrppd6 ◽

2021 ◽

Author(s):

Inswasti Cahyani ◽

John Tyson ◽

Nadine Holmes ◽

Josh Quick ◽

Nicholas Loman ◽

...

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Dna Extraction ◽

Human Cell ◽

Glass Bead ◽

Human Cell Lines ◽

Library Preparation ◽

Long Read ◽

Full Protocol ◽

Ultra High Molecular Weight

This collection of protocols is designed to enable ultra-long (UL) reads on Nanopore sequencers. It is split into five sections dealing with ultra-high molecular weight (UHMW) DNA: Extraction QC Library preparation Nemo clean-up using glass beads and Hexamminecobalt(III) Chloride, aka. CoHex. Flowcell priming and library loading We have tested and optimised the full protocol in human cell lines. Various options are available for each of the steps and we hope that the components here will be useful to the community and provide a long read toolkit.

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Evaluation of Two Extraction Methods for the Analysis of Hydrophilic Low Molecular Weight Compounds from Ganoderma lucidum Spores and Antiproliferative Activity on Human Cell Lines

Applied Sciences ◽

10.3390/app10114033 ◽

2020 ◽

Vol 10 (11) ◽

pp. 4033

Author(s):

Maria Michela Salvatore ◽

Vincenza De Gregorio ◽

Monica Gallo ◽

Maria Michela Corsaro ◽

Angela Casillo ◽

...

Keyword(s):

Molecular Weight ◽

Cell Lines ◽

Human Cell ◽

Ganoderma Lucidum ◽

Polar Compounds ◽

Extraction Methods ◽

Hot Water ◽

Aqueous Extracts ◽

Human Cell Lines ◽

Soxhlet Apparatus

Background: The genus Ganoderma includes about 80 species of mushrooms. Ganoderma lucidum is the best-known fungal species in mycotherapy and likely has the highest number of studies. Numerous bioactive compounds seem to be responsible for its beneficial effects; in particular, triterpenes, peptidoglycans and polysaccharides are the main physiologically active constituents. The aim of the present work is to identify the main bioactive components in aqueous extracts of G. lucidum spores obtained by two different extraction processes. Methods: The spores were at first extracted by a Soxhlet apparatus with n-hexane and the aqueous solutions were submitted to two different procedures: hot water extraction (HWE) and rapid solid-liquid dynamic extraction (RSLDE) using the Naviglio extractor. The extracts were then dialyzed to separate the compounds with higher molecular weight from polar compounds with lower molecular weight. The aqueous extracts and dialyzed fractions were tested on two human cell lines: human colonic epithelial cells (Caco-2) and human colorectal carcinoma cells (HTC-116). Results: GC-MS and NMR data revealed the presence of a mixture of glucose and mannitol in ratio 3.7:1 in the most active fraction. Conclusions: The outside dialysis phase of RSLDE extract seems to be particularly cytotoxic for HTC-116 and, interestingly, solutions with different concentrations of pure mannitol showed cytotoxic activity against this cell line too, although to a lesser extent.

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