Global translation profiling during red blood cell development. (A) Workflow for parallel ribosome and RNA profiling during erythropoiesis. (B) Ribosome footprints (RFPs) delineate known CDSs. Metagene plot shows the rise and fall in 28-nt RFP density (reads per million mapped reads, RPM) near starts and ends of annotated CDSs, respectively. The 12-nt and 15-nt offsets from starts and ends reflect distances from RFP 5′ termini to the ribosome P- and A-site codons at translation initiation and termination, respectively (see inset). (C) Subcodon resolution of ribosome footprints. Note that 3-nt codon periodicity relative to the known CDS is seen for 28-nt RFPs but not RNA-seq reads. (D) Ribosome footprints are highly specific to coding regions. Boxplots show density of Ribo-seq and RNA-seq reads at UTRs or introns relative to that of the associated CDS. (E) Ribosome and RNA profiling of the locus encoding the β-globin–major chain (Hbb-b1). Tracks display Ribo-seq and RNA-seq signal as density of mapped strand-specific reads. The gene model with CDS (blue) and UTR (green) regions is shown at the bottom. Kb, kilobases; RNase I, ribonuclease I.