Experimental Setup and Data Analysis Considerations for DNA- and RNA-SIP Experiments in the Omics Era
Language English Country United States Media print
Document type Journal Article, Research Support, Non-U.S. Gov't
- Keywords
- Amplicon sequencing, DNA-SIP, Network analysis, Omics, RNA-SIP,
- MeSH
- Data Analysis MeSH
- RNA, Bacterial chemistry MeSH
- Deuterium analysis MeSH
- DNA, Bacterial chemistry MeSH
- DNA metabolism MeSH
- Isotope Labeling methods MeSH
- Nitrogen Isotopes analysis MeSH
- Oxygen Isotopes analysis MeSH
- Carbon Isotopes analysis MeSH
- RNA metabolism MeSH
- High-Throughput Nucleotide Sequencing MeSH
- Research Design MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- RNA, Bacterial MeSH
- Deuterium MeSH
- DNA, Bacterial MeSH
- DNA MeSH
- Nitrogen Isotopes MeSH
- Oxygen Isotopes MeSH
- Carbon Isotopes MeSH
- RNA MeSH
Careful and thoughtful experimental design is crucial to the success of any SIP experiment. This chapter discusses the essential aspects of designing a SIP experiment, focusing primarily on DNA- and RNA-SIP. The design aspects discussed here begin with considerations for carrying out the incubation, such as, the effect of choosing different stable isotopes and target biomolecules, to what degree should a labeled substrate be enriched, what concentration to use, and how long should the incubation take. Then tips and pitfalls in the technical execution of SIP are listed, including how much nucleic acids should be loaded, how many fractions to collect, and what centrifuge rotor to use. Lastly, a brief overview of the current methods for analyzing SIP data is presented, focusing on high-throughput amplicon sequencing, together with a discussion on how the choice of analysis method might affect the experimental design.
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