Reproducibility and consistency of in vitro nucleosome reconstitutions demonstrated by invitrosome isolation and sequencing

Nucleosomes and their positions in the eukaryotic genome play an important role in regulating gene expression by influencing accessibility to DNA. Many factors influence a nucleosome's final position in the chromatin landscape including the underlying genomic sequence. One of the primary reason...

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Bibliographic Details
Published in:PloS one 2014-08, Vol.9 (8), p.e103752-e103752
Main Authors: Kempton, Colton E, Heninger, Justin R, Johnson, Steven M
Format: Article
Language:English
Subjects:
Animals
Biology and Life Sciences
Cell Fractionation - methods
Chromatin
Chromatin Assembly and Disassembly
Deoxyribonucleic acid
DNA
DNA - analysis
DNA - metabolism
Experiments
Gene expression
Gene sequencing
Genomes
In Vitro Techniques
Influence
Molecular biology
Nucleosomes
Nucleosomes - chemistry
Nucleosomes - genetics
Nucleosomes - metabolism
Nucleotide sequence
Plasmids
Plasmids - chemistry
Plasmids - genetics
Reproducibility
Reproducibility of Results
Research and Analysis Methods
Sequence Analysis, DNA
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Summary:Nucleosomes and their positions in the eukaryotic genome play an important role in regulating gene expression by influencing accessibility to DNA. Many factors influence a nucleosome's final position in the chromatin landscape including the underlying genomic sequence. One of the primary reasons for performing in vitro nucleosome reconstitution experiments is to identify how the underlying DNA sequence will influence a nucleosome's position in the absence of other compounding cellular factors. However, concerns have been raised about the reproducibility of data generated from these kinds of experiments. Here we present data for in vitro nucleosome reconstitution experiments performed on linear plasmid DNA that demonstrate that, when coverage is deep enough, these reconstitution experiments are exquisitely reproducible and highly consistent. Our data also suggests that a coverage depth of 35X be maintained for maximal confidence when assaying nucleosome positions, but lower coverage levels may be generally sufficient. These coverage depth recommendations are sufficient in the experimental system and conditions used in this study, but may vary depending on the exact parameters used in other systems.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0103752