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Purification of micrococcal nuclease (MNase) for use in ribosome profiling of high-salinity extremophiles

ABSTRACT: Purification of micrococcal nuclease (MNase) for use in ribosome profiling of high-salinity extremophiles

PROVIDER: PRJNA1123824 | ENA |

REPOSITORIES: ENA

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Purification of micrococcal nuclease (MNase) for use in ribosome profiling of high-salinity extremophiles

Project description:Nucleases, i.e. enzymes that catalyze the hydrolysis of phosphodiester bonds in nucleic acids, are essential tools in molecular biology and biotechnology. Staphylococcus aureus nuclease (MNase) is particularly interesting due to its thermostability and Ca2+-dependence, making it the prime choice for applications where nuclease modulation is critical, such as ribosome profiling in bacteria and halophilic archaea. The latter poses a technical and economical challenge, as high salt reaction conditions negatively impact MNase activity, necessitating large amounts of nuclease to be used for achieving efficient cleavage. Here, we set out to generate an optimized production protocol for two forms of MNase — fully processed MNaseA and the 19 aa propeptide containing MNaseB — and to biochemically benchmark them against a commercial nuclease. Our results show that both MNases are highly active in normal reaction conditions, but MNaseA maintains higher enzymatic activity in high salt concentrations than MNaseB. MNaseA also retains >90% of its activity after multiple freeze-thaw cycles when stored at -80°C in a buffer containing 5% glycerol. Importantly, ribosome profiling experiments in Haloferax volcanii demonstrated that MNaseA produces ribosome footprints highly comparable to those obtained with the commercial nuclease, making it a suitable alternative for high-salt ribosome profiling applications. In conclusion, our method can be easily implemented for efficient MNaseA production, thereby providing access to an effective, robust, and cost-efficient alternative to commercial nucleases, as well as facilitating future translation studies into halophilic organisms.

2024-06-22 | GSE269836 | GEO

Nucleosome position mapping by micrococcal nuclease analysis of S. cerevisiae cells in raffinose and galactose containing media

Project description:We analyed the nucleosome positions by using 2 concentrations of micrococcal nuclease of yeast strains that were grown in raffinose and galactose containing media (synthetic complete).

2019-05-14 | GSE116337 | GEO

Micrococcal nuclease sequencing analysis for nucleosome localization of mouse fetal liver erythroblasts

Project description:Nucleosomal DNA was prepared using Simple ChIP Enzymatic Chromatin IP Kit according to manufacturer’s instruction. Briefly, Nuclei were isolated from purified Ter119 negative or in vitro cultured erythroblasts. Cross-linked native chromatin was then digested with MNase into mononucleosomal DNA. Sequencing libraries were generated from nucleosomal DNA, and sequencing was carried out using the Illumina system according to the manufacturer’s specification. In this study, we purified chromatin from in vitro cultured mouse fetal liver erythroblasts on day 0, day 1, and day 2. The chromatins were digested by micrococcal nuclease to make mononucleosomal products, which were further analyzed by next generation sequencing analysis. We aim to determine the dynamic changes of nucleosome during terminal erythropoiesis.

2014-10-01 | E-GEOD-55825 | biostudies-arrayexpress

Nucleosome position mapping by micrococcal nuclease analysis in S. cerevisiae of Top1-AID/Top2-AID strains with auxin in galactose-rich media

Project description:We analyed the nucleosome positions by using 2 concentrations of micrococcal nuclease of haploid yeast strains that were grown in galactose containing synthetic complete media. Strains contained AID-tags at the endogeous TOP1 and TOP2 genes. One strain contained OsTIR1 and these cells were either untreated or treated for 60 min with 500 uM auxin.

2023-04-28 | GSE196945 | GEO

EXTREMOPHILES

Project description:International Space Station flight project EXTREMOPHILES.

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Nucleosome position mapping by micrococcal nuclease analysis of S. cerevisiae anchor-away Sth1, Swi2 and/of TBP cells in raffinose and galactose-rich media in rapamycin or DMSO

Project description:We analyed the nucleosome positions by using 2 concentrations of micrococcal nuclease on yeast strains that were grown in raffinose or galactose containing media (synthetic complete). Strains contained FRB-tags to Sth1, Swi2, Sth1 & Swi2 or Sth1 and TBP, where TBP was tagged on one of two alleles in a diploid strain. Cells were treated for 60 min either with 7.5 uM rapamycin or the same volume of DMSO.

2023-01-11 | GSE190737 | GEO

Tissue-specific nuclei purification from animal models for genome-wide expression and chromatin profiling.

Project description:Here we describe a method for purifying nuclei from specific tissues of animal models that allows simultaneous analysis of both expression and chromatin profiles. The method is based on in vivo labeling of the nuclear envelope and subsequent affinity purification. We describe the use of the method to isolate nuclei from muscle. We determined genes preferentially expressed in muscle cells of adult C. elegans by analyzing RNA from total and affinity-puridied muscle nuclei using microarrays. We also determined the nucleosome occupancy in these nuclei by micrococcal nuclease mapping and paired-end sequencing.

2011-12-05 | E-GEOD-31602 | biostudies-arrayexpress

MNase-seq analysis of wild type and asf1 mutant of Sordaria macrospora

Project description:The aim of this analysis was to analyze nucleosome distribution in the filamentous ascomycete Sordaria macrospora by micrococcal nuclease digestion and sequencing.

2018-11-26 | GSE92461 | GEO

Differential features of lamina-associated domains mapped by ChIP-sequencing from sonicated or micrococcal nuclease-digested chromatin

Project description:The nuclear lamina interacts with the genome through megabase-size lamina-associated domains (LADs). LADs have been identified in proximity labeling assays and recently by chromatin immunoprecipitation-sequencing (ChIP-seq) of A- and B-type lamins. LADs localize mainly to the nuclear periphery, they are gene-poor and largely heterochromatic. Here, we show that the mode of chromatin fragmentation for ChIP, namely either bath sonication (used to date for ChIP of nuclear lamins) or digestion with micrococcal nuclease (MNase) leads to the discovery of distinct sets of lamin-interacting domains (which we refer to as LiDs) with distinct gene content, histone composition enrichment and relationship to lamin B1-interacting domains. We find that total genome coverage by lamin A/C ('LMNA') LiDs identified in sonicated or MNase-digested chromatin is similar (~730 megabases). Over half of these domains, however, are uniquely detected in sonicated or MNase-digested chromatin. Whereas sonication-specific LMNA LiDs are gene-poor and devoid of a broad panel of histone modifications, MNase-specific LMNA LiDs are of higher gene density and are enriched in H3K9me3, H3K27me3 and in histone variant H2A.Z. Analysis of published LMNB1 LADs and of LMNB1 LiDs identified by ChIP-seq further shows that LMNA can associate with 'open' chromatin domains displaying euchromatin features which are not associated with LMNB1. The differential genetic and epigenetic properties of lamin-interacting chromatin domains indicate the existence of distinct LiD populations identifiable in different chromatin contexts, including nuclease-accessible 'open' regions presumably localized in the nuclear interior.

2015-03-24 | GSE57149 | GEO

The Drosophila BEAF Insulator Protein Interacts with the Polybromo Subunit of the PBAP Chromatin Remodeling Complex [Mnase-seq]

Project description:We examine the effect of this interaction on gene expression and chromatin structure using precision run-on sequencing (PRO-seq) and micrococcal nuclease sequencing (MNase-seq) after RNAi-mediated knockdown in cultured S2 cells. We examine the effect of the interaction between BEAF and polybromo on gene expression and chromatin structure using precision run-on sequencing (PRO-seq) and micrococcal nuclease sequencing (MNase-seq) after RNAi-mediated knockdown in cultured S2 cells.

2022-03-05 | GSE197574 | GEO

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