Experimental Setup and Data Analysis Considerations for DNA- and RNA-SIP Experiments in the Omics Era
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.
Key wordsDNA-SIP RNA-SIP Amplicon sequencing Omics Network analysis
The manuscript for this chapter was written online using authors. R.A. was supported by BC CAS, ISB & SoWa RI (MEYS; projects LM2015075, EF16_013/0001782—SoWa Ecosystems Research).
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