A custom library construction method for super-resolution ribosome profiling in Arabidopsis

Ribosome profiling (aka Ribo-seq) is the deep sequencing of ribosome footprints (RFs). It maps and quantifies ribosome occupancy on mRNA, which enables the identification of coding regions and the accurate quantification of translation efficiency. We previously optimized the Ribo-seq method in Arabidopsis and tomato (Hsu et al., 2016; Wu et al., 2019; Wu and Hsu, 2022) to obtain precise RFs with strong 3-nucleotide periodicity, a feature displayed by actively translating ribosomes and a benchmark of high-quality Ribo-seq (Brar and Weissman, 2015). This strong periodicity allowed us to confidently define numerous unannotated translation events across plants (Hsu et al., 2016; Wu et al., 2019; Wu and Hsu, 2022). Recently, several key commercial reagents used in our methods were discontinued; thus, there is an urgent need to develop a new protocol. Here, we report an updated protocol that adapts two custom library construction methods (McGlincy and Ingolia, 2017; Li et al., 2021) for plants. We applied this new protocol to Arabidopsis seedlings and obtained high-quality data. We describe our step-by-step method and discuss crucial considerations for Ribo-seq experiments. We also provide a bioinformatic pipeline to perform essential quality control analyses on Ribo-seq data. Our approach should be readily applicable to other plant species with minimal modifications.