(A) Digestion of polysomes with RNase I degrades ribosomes. A lysate was made from S2 cells using a previous version of our protocol. Aliquots of this lysate were digested with increasing amounts of RNase I, and resolved on 10–50% sucrose gradients. As amounts of RNase I increase, the heights of all peaks—including the monosomal (80S) peak—decrease before polysomes are fully resolved to monosomes. (B) as in (A), but using micrococcal nuclease (MNase) and our current protocol. From 0.5 to 2 U MNase/μg total RNA, monosomes are resolved with no reduction in the size of the monosome peak. This result indicates that Drosophila ribosomes are stable to MNase over a broad range of concentrations, whereas the mRNA between ribosomes is digested. (C) Ribosome protection assay. A 320 nucleotide fragment of enolase (FlyBase accession: FBgn0000579) was amplified using oligos oJGD123 & oJGD124 (Supplementary file 2). A body-labeled probe against this sequence was transcribed from this template using α32P-UTP and the T7 MaxiScript kit (Ambion). S2 cell lysates were prepared as in methods and aliquoted. Aliquots were digested as in methods, except with 0, 0.5, 1, 2, 3 or 4 U MNase/μg total RNA. Monosomes were sedimented through a sucrose cushion, resuspended in 600 μl 10 mM Tris pH 7.0, and their RNAs extracted as in ‘Materials and methods’. Concentrations were determined using a NanoDrop spectrophotometer. 5 μg of each sample was hybridized to 50,000 CPM of probe overnight at 42°C. Single-stranded regions were digested with RNase A/T1 and the remaining footprint: probe duplexes detected using the mirVana micro-RNA detection kit (Ambion), resolved on a 15% TBE-urea gel (Invitrogen), and visualized on a Storm phosphorimager (Molecular Dynamics by GE Healthcare Bio-Sciences, Pittsburgh, PA). For size markers, we end-labeled the Novex 10 bp dsDNA ladder (Invitrogen) with 32P. Over two-fold range of nuclease concentrations, the ∼30 nt peak corresponding to ribosome-protected footprints remains constant in size and intensity, indicating a lack of degradation consistent with the unchanged monosome peak height across this range of digestion conditions in (B). Also visible is a roughly 60 nt band which we infer to be protected by adjacent ribosomes (disomes) that sterically exclude the nuclease. This interpretation is consistent with the presence of a small disome peak in digested samples (c.f. panels B and D, and Figure 1A). (D) A polysome lysate was prepared from S2 cells and resolved in 10–50% sucrose gradients, with or without prior digestion with 3 U MNase/μg total RNA (E) A culture of S2 cells was split into aliquots and processed using our current protocol as if they were independent samples. Total counts aligning to the coding region of each gene were tabulated in each replicate. Genes sharing at least 128 footprint counts between replicates (red) are well-correlated, demonstrating the assay is robust (see full discussion in Figure 1—figure supplement 2). Source data may be found in supplementary table 1 (at Dryad: Dunn et al., 2013).