Fig. 1. Next-generation sequencing based methods for global analysis of translation. (A) In ribo-seq, ribosome-bound mRNAs are purified by sedimentation following cell lysis. Nuclease treatment is then used to degrade unmasked RNA sections, leaving 20–30 nucleotide-long ribosome protected fragments known as ribosome footprints. The footprints are then subjected to library preparation and next-generation sequencing (below). The read densities can be used to both distinguish individual ORFs, as well as to quantify their translation rates, with a distinctive three-nucleotide periodic footprint pattern that is indicative of ribosome translocation often visible throughout the length of the ORF. (B) TRAP-seq differs from ribo-seq in the way ribosomes are purified. Rather than sedimentation, epitope tagged ribosomal proteins which are either stably expressed in cultured cells or transgenically in a given cell-type of an in vivo model, are subjected to immunoprecipitation in order to pulldown the translating ribosomes. Nuclease treatment is then used to degrade unmasked RNA sections, followed by library preparation, and next-generation sequencing of the footprints as before (below). (C) Proximity-specific ribo-seq allows assessment of subcellularly localized translation by tagging ribosomal proteins with a biotin acceptor peptide (Avi), coupled with expression of a subcellularly localized biotin ligase (BirA). A carefully optimized biotin pulse is then applied in order to induce biotinylation of the Avi tagged ribosomes in close proximity of the BirA. Subsequently, cells are lysed and biotinylated ribosomes are affinity purified using streptavidin conjugated beads, followed by nuclease treatment, library preparation, and next-generation sequencing of the ribosome footprints as before (below).