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, how enriched should a labelled substrate be, what concentration to use and how long the incubation should 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 analysing 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.
Nitrogen fixation and assimilation processes are vital to the functioning of any ecosystem. Nevertheless, studying these processes using ¹⁵N-based stable isotope probing was so far limited because of technical challenges related to the relative rarity of nitrogen in nucleic acids and proteins compared to carbon, and because of its absence in lipids. However, the recent adoption of high-throughput sequencing and statistical modelling methods to SIP studies increased the sensitivity of the method and enabled overcoming some of the challenges. This chapter describes in detail how to perform DNA- and RNA-SIP using ¹⁵N.