An optimised Chromatin Immunoprecipitation (ChIP) method for starchy leaves of Nicotiana benthamiana to study histone modifications of an allotetraploid plant

Abstract Background All flowering plants have evolved through multiple rounds of polyploidy throughout the evolutionary process. Intergenomic interactions between subgenomes in polyploid plants are predicted to induce chromatin modifications such as histone modifications to regulate expression of gene homoeologs. Nicotiana benthamiana is an ancient allotetraploid plant with ecotypes collected from climatically diverse regions of Australia. Studying the differences in chromatin landscape of this unique collection will shed light on the importance of chromatin modifications in gene regulation in polyploids as well its implications in adaptation of plants in environmentally diverse conditions. N.benthamiana is also an important biotechnological tool and it is widely used in virological research and functional genomics. Chromatin Immunoprecipitation and high throughput DNA sequencing (ChIP-seq) is well established technique used to study histone modifications. However, due to the starchy nature of mature N.benthamiana leaves, previously published protocols were unsuitable. The aim of this study was to optimise ChIP protocol for N.benthamiana leaves to facilitate comparison of chromatin modifications in two closely related ecotypes. Results Several steps of ChIP were optimised including tissue harvesting, nuclei isolation, nuclei storage, DNA shearing and DNA recovery. The higher amounts of starch in mature N.benthamiana leaves that co-precipitated with nuclei using previously published protocols, hindered chromatin shearing and resulted in low recovery of ChIP DNA. The optimised method reduced starch contamination and resulted in isolation of high quality nuclei suitable for next generation sequencing. Commonly available antibodies targeting histone 3 lysine 4 trimethylation (H3K4me3) and histone 3 lysine 9 dimethylation (H3K9me2) histone modifications were used and success of ChIP was confirmed by PCR and next generation sequencing. Conclusions An optimised ChIP method for mature leaves of N.benthamiana is described. It is relatively less laborious than previously published protocols and allows extraction of high quality nuclear genomic DNA from N.benthamiana. It is also the first comprehensive ChIP method for starchy leaves of N.benthamiana suitable for preparation of ChIP libraries for next generation sequencing.

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An optimised chromatin immunoprecipitation (ChIP) method for starchy leaves of Nicotiana benthamiana to study histone modifications of an allotetraploid plant

Molecular Biology Reports ◽

10.1007/s11033-020-06013-1 ◽

2020 ◽

Vol 47 (12) ◽

pp. 9499-9509

Author(s):

Buddhini Ranawaka ◽

Milos Tanurdzic ◽

Peter Waterhouse ◽

Fatima Naim

Keyword(s):

Histone Modifications ◽

Chromatin Immunoprecipitation ◽

Nicotiana Benthamiana ◽

Evolutionary Process ◽

Downstream Processing ◽

Chromatin Modifications ◽

Dna Recovery ◽

Histone 3 ◽

Wide Scale ◽

Intergenomic Interactions

AbstractAll flowering plants have evolved through multiple rounds of polyploidy throughout the evolutionary process. Intergenomic interactions between subgenomes in polyploid plants are predicted to induce chromatin modifications such as histone modifications to regulate expression of gene homoeologs. Nicotiana benthamiana is an ancient allotetraploid plant with ecotypes collected from climatically diverse regions of Australia. Studying the chromatin landscape of this unique collection will likely shed light on the importance of chromatin modifications in gene regulation in polyploids as well its implications in adaptation of plants in environmentally diverse conditions. Generally, chromatin immunoprecipitation and high throughput DNA sequencing (ChIP-seq) is used to study chromatin modifications. However, due to the starchy nature of mature N. benthamiana leaves, previously published protocols were unsuitable. The higher amounts of starch in leaves that co-precipitated with nuclei hindered downstream processing of DNA. Here we present an optimised ChIP protocol for N. benthamiana leaves to facilitate comparison of chromatin modifications in two closely related ecotypes. Several steps of ChIP were optimised including tissue harvesting, nuclei isolation, nuclei storage, DNA shearing and DNA recovery. Commonly available antibodies targeting histone 3 lysine 4 trimethylation (H3K4me3) and histone 3 lysine 9 dimethylation (H3K9me2) histone modifications were used and success of ChIP was confirmed by PCR and next generation sequencing. Collectively, our optimised method is the first comprehensive ChIP method for mature starchy leaves of N. benthamiana to enable studies of chromatin landscape at the genome-wide scale.

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Automated Workflow for Somatic and Germline Next Generation Sequencing Analysis in Routine Clinical Cancer Diagnostics

Cancers ◽

10.3390/cancers11111691 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1691

Author(s):

Muscarella ◽

Fabrizio ◽

De Bonis ◽

Mancini ◽

Balsamo ◽

...

Keyword(s):

Next Generation Sequencing ◽

Scale Up ◽

Cancer Diagnostics ◽

Sequencing Analysis ◽

Library Preparation ◽

Next Generation ◽

High Quality ◽

High Coverage ◽

Dna Recovery ◽

Generation Sequencing

Thanks to personalized medicine trends and collaborations between industry, clinical research groups and regulatory agencies, next generation sequencing (NGS) is turning into a common practice faster than one could have originally expected. When considering clinical applications of NGS in oncology, a rapid workflow for DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissue samples, as well as producing high quality library preparation, can be real challenges. Here we consider these targets and how applying effective automation technology to NGS workflows may help improve yield, timing and quality-control. We firstly evaluated DNA recovery from archived FFPE blocks from three different manual extraction methods and two automated extraction workstations. The workflow was then implemented to somatic (lung/colon panel) and germline (BRCA1/2) library preparation for NGS analysis exploiting two automated workstations. All commercial kits gave good results in terms of DNA yield and quality. On the other hand, the automated workstation workflow has been proven to be a valid automatic extraction system to obtain high quality DNA suitable for NGS analysis (lung/colon Ampli-seq panel). Moreover, it can be efficiently integrated with an open liquid handling platform to provide high-quality libraries from germline DNA with more reproducibility and high coverage for targeted sequences in less time (BRCA1/2). The introduction of automation in routine workflow leads to an improvement of NGS standardization and increased scale up of sample preparations, reducing labor and timing, with optimization of reagents and management.

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Optimizing preservation protocols to extract high‐quality RNA from different tissues of echinoderms for next‐generation sequencing

Molecular Ecology Resources ◽

10.1111/1755-0998.12122 ◽

2013 ◽

Vol 13 (5) ◽

pp. 884-889 ◽

Cited By ~ 12

Author(s):

Rocío Pérez‐Portela ◽

Ana Riesgo

Keyword(s):

Next Generation Sequencing ◽

Next Generation ◽

High Quality ◽

Generation Sequencing ◽

Different Tissues

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A Rapid, High-Quality, Cost-Effective, Comprehensive and Expandable Targeted Next-Generation Sequencing Assay for Inherited Heart Diseases

Circulation Research ◽

10.1161/circresaha.115.306723 ◽

2015 ◽

Vol 117 (7) ◽

pp. 603-611 ◽

Cited By ~ 20

Author(s):

Kitchener D. Wilson ◽

Peidong Shen ◽

Eula Fung ◽

Ioannis Karakikes ◽

Angela Zhang ◽

...

Keyword(s):

Next Generation Sequencing ◽

Heart Diseases ◽

Cost Effective ◽

Next Generation ◽

High Quality ◽

Quality Cost ◽

Targeted Next Generation Sequencing ◽

Generation Sequencing

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Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis of the Penicillium chrysogenum Velvet Protein PcVelA Identifies Methyltransferase PcLlmA as a Novel Downstream Regulator of Fungal Development

mSphere ◽

10.1128/msphere.00149-16 ◽

2016 ◽

Vol 1 (4) ◽

Cited By ~ 7

Author(s):

Kordula Becker ◽

Sandra Ziemons ◽

Katharina Lentz ◽

Michael Freitag ◽

Ulrich Kück

Keyword(s):

Next Generation Sequencing ◽

Secondary Metabolism ◽

Filamentous Fungi ◽

Chromatin Immunoprecipitation ◽

Next Generation ◽

Content Type ◽

Major Interest ◽

Lactam Antibiotic ◽

Velvet Complex ◽

Generation Sequencing

ABSTRACT Filamentous fungi are of major interest for biotechnological and pharmaceutical applications. This is due mainly to their ability to produce a wide variety of secondary metabolites, many of which are relevant as antibiotics. One of the most prominent examples is penicillin, a β-lactam antibiotic that is produced on the industrial scale by fermentation of P. chrysogenum. In recent years, the multisubunit protein complex velvet has been identified as one of the key regulators of fungal secondary metabolism and development. However, until recently, only a little has been known about how velvet mediates regulation at the molecular level. To address this issue, we performed ChIP-seq (chromatin immunoprecipitation in combination with next-generation sequencing) on and follow-up analysis of PcVelA, the core component of the velvet complex in P. chrysogenum. We demonstrate direct involvement of velvet in transcriptional control and present the putative methyltransferase PcLlmA as a new downstream factor and interaction partner of PcVelA. Penicillium chrysogenum is the sole industrial producer of the β-lactam antibiotic penicillin, which is the most commonly used drug for treating bacterial infections. In P. chrysogenum and other filamentous fungi, secondary metabolism and morphogenesis are controlled by the highly conserved multisubunit velvet complex. Here we present the first chromatin immunoprecipitation next-generation sequencing (ChIP-seq) analysis of a fungal velvet protein, providing experimental evidence that a velvet homologue in P. chrysogenum (PcVelA) acts as a direct transcriptional regulator at the DNA level in addition to functioning as a regulator at the protein level in P. chrysogenum, which was previously described. We identified many target genes that are related to processes known to be dependent on PcVelA, e.g., secondary metabolism as well as asexual and sexual development. We also identified seven PcVelA target genes that encode putative methyltransferases. Yeast two-hybrid and bimolecular fluorescence complementation analyses showed that one of the putative methyltransferases, PcLlmA, directly interacts with PcVelA. Furthermore, functional characterization of PcLlmA demonstrated that this protein is involved in the regulation of conidiosporogenesis, pellet formation, and hyphal morphology, all traits with major biotechnological relevance. IMPORTANCE Filamentous fungi are of major interest for biotechnological and pharmaceutical applications. This is due mainly to their ability to produce a wide variety of secondary metabolites, many of which are relevant as antibiotics. One of the most prominent examples is penicillin, a β-lactam antibiotic that is produced on the industrial scale by fermentation of P. chrysogenum. In recent years, the multisubunit protein complex velvet has been identified as one of the key regulators of fungal secondary metabolism and development. However, until recently, only a little has been known about how velvet mediates regulation at the molecular level. To address this issue, we performed ChIP-seq (chromatin immunoprecipitation in combination with next-generation sequencing) on and follow-up analysis of PcVelA, the core component of the velvet complex in P. chrysogenum. We demonstrate direct involvement of velvet in transcriptional control and present the putative methyltransferase PcLlmA as a new downstream factor and interaction partner of PcVelA.

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