Transcriptome-wide measurement of ribosomal occupancy by ribosome profiling

Experiments for optimization of the ribosome profiling protocol using C. elegans lysates. We report that optimizing the cutting accuracy to a narrow window of 28-30 nucleotides when PAGE-purifying the ribosome-protected fragments (RPFs) significantly improves the quality of the RPF library. In addition, we find that purifying monosomes by sucrose gradient fractionation clearly removed more contaminating ribosomal RNA from the samples compared to the purification by gel filtration columns. Overall design: Experiment 1 (samples 1-4): Comparison between different methods to isolate monosomes and between different library preparations. Experiment 2 (samples 5-8): Optimization of the cutting accuracy to PAGE purify ribosome-protected fragments.