Fig. S1.

Technical validation of ribosome profiling experiments. (A) Simplified representation of the modified ribosome profiling method. RNase I digestion leaves a 5′-OH and a 3′-cyclophosphate. T4 polynucleotide kinase treatment performed in the absence of ATP converts the 3′-cyclophosphate into a 3′-OH whereas phosphorylating the 5′-OH ends in presence of ATP. Libraries are then generated as described in Materials and Methods. (B) Example of 260-nm optical density profile from sucrose gradient fractions. RNase I-digested lysates (red line) harbor clear monosome enrichment, whereas untreated controls (dark dashes) exhibit a standard polysome-enriched profile. Monosomal RNA was then extracted from fractions delimited with a red bar. (C) Extracted RNAs were resolved on 15% TBE-urea-PAGEs to select 28- to 36-nt-long RNA fragments. (D) Homogenous size distribution of libraries with a strong and sharp library peak at the expected size (∼150 bp). (E) Technical reproducibility of technical replicates for each individual gene (log2 RPKM). R² indicates Pearson correlation at the top of each panel. Four samples (two per time point) were treated as described before. Then, fragmented RNAs were split, resolved on three distinct 15% TBE-urea-PAGEs, and analyzed as described in Materials and Methods.