Ribosome profiling sample preparation and library preparation

Ribosome footprint purification and subsequent sequencing of ribosome footprints was performed with RiboLace Module 1 and LACEseq (IMMAGINA Biotechnology). Active ribosomes were isolated using the RiboLace kit according to the manufacturer’s instructions. Flash frozen tissue samples were ground with a mortar and pestle in liquid nitrogen. The resulting powder was resuspended in 800 μL of lysis buffer (final concentration of 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 100 μg/μL cycloheximide, 1% Triton X-100, and 25 U/mL DNase I). After centrifugation at 20,000 g for 5 min, the supernatant was transferred to a new tube and kept on ice for 20 min. Ribosome footprints were generated using a nuclease digestion and ribosome footprints were captured using functionalized Ribolace beads, which were then purified using acid phenol: chloroform according to the manufacturer’s instructions. Purified samples were run on a 15% TBE-Urea polyacrylamide gel, stained using SYBR Gold, and regions corresponding to 25 to 35 nt were size selected. The gel slices were sheared, suspended in elution buffer, incubated at −80°C for 60 min and then incubated at room temperature on a standing rotator overnight. Samples were isopropanol precipitated with glycoblue and suspended in 11 μL of TR buffer. Concentration of the RPFs was assessed using the Qubit microRNA Assay Kit (Thermo Fisher).

Library preparation of purified ribosome footprints was performed using LACEseq. 5′ phosphorylation, linker ligation, circularization, reverse transcription and PCR amplification were all done according to the manufacturer’s instructions. LACEseq library preparation involves 2 rounds of PCR amplification, in which the second round incorporates a unique dual index (UDI) (IMMAGINA) for each sample. Libraries were run on a 6% TBE polyacrylamide gel, stained with SYBR Gold, and regions approximately 200 nt in size were selected. The quality and quantity of the libraries was assessed using the high-sensitivity DNA chip on the Bioanalyzer (Agilent). Libraries were pooled and sequenced in single end mode on the Illumina NextSeq 500 System High Output kit (Illumina) by the OSU Genomics and Proteomics Center and also on the Illumina NovaSeq 6000 System S1 flow cell by the Michigan State University Genomics core lab.