In the past 10 years, standard transcriptome sequencing protocols were optimized so well that no prior experience is required to prepare the sequencing library. Often, all enzymatic steps are designed to work in the same reaction tube minimizing handling time and reducing human errors. Ribosome profiling stands out from these methods. It is a very demanding technique that requires isolation of intact ribosomes, and thus there are multiple additional considerations that must be accounted for (McGlincy and Ingolia, Methods 126:112-129, 2017). In this chapter, we discuss how to select a ribonuclease to produce ribosomal footprints that will be later converted to the sequencing library. Several ribonucleases with different cutting patterns are commercially available. Selecting the right one for the experimental application can save a lot of time and frustration.
Keywords: Ribonuclease; Ribosome profiling; Translation; rRNA.