title: Ribo-Seq of P. putida KT2440
design: The preparation of Ribo-Seq samples utilized a protocol adapted from previously described methods designed for axenic bacterial cultures. Briefly, the bacterial cultures were treated with chloramphenicol and pelleted. At this point, we modified this protocol to address the chloramphenicol-resistance of Pseudomonas putida KT2440 by resuspending the pellet in RNAlater, and flash-freezing in liquid nitrogen. Samples were thawed, pelleted, and RNAlater removed, before proceeding to mechanical bacterial lysis. Bacterial lysis was performed in the presence of a lysis solution containing additional chloramphenicol and Guanosine-5-[(,)-imido]triphosphate (GMPPNP) to inhibit protein elongation. Lysates were treated with MNase and DNase to digest nucleic acids that were not protected by ribosomes. Monosomes were recovered using RNeasy mini spin size-exclusion columns (Qiagen) and RNA Clean & Concentrator-5 kit (Zymo). rRNA was removed with the QIAseq FastSelect-5S/16S/23S kit (Qiagen), and MetaRibo-Seq libraries were constructed using the NEBNext Small RNA Library Prep set for Illumina. Amplification was followed in real-time with SYBR-Green and stopped upon plateau plateau, and PCR products were purified with the Select-a-size DNA Clear & Concentrator kit (Zymo).