Monosome-seq, disome-seq, and mRNA-seq

Libraries were prepared as described previously [17, 24, 55], with modifications. Fifty thousand units (A₂₆₀) of ribosome dissolved in the PLB buffer were treated with 750 U RNase I (Ambion, AM2294) at 25 °C for 2 h. Our pilot experiments, as well as previous studies, have shown that sucrose gradient centrifugation is not necessary [55, 71–73] as long as the digestion with RNase I is complete (Additional file 1: Fig. S1); rather, this step consumes a large amount of ribosome (especially disome) samples. The following-up computational analyses can help to tell if the RNA fragments being collected are largely protected by ribosomes: whether the fragments are restricted to the coding sequences, whether the fragments are enriched in certain lengths, and whether a 3-nt periodicity exists (Additional file 1: Fig. S3c-f). Therefore, RNA was directly extracted from the solution for RNase I digestion with hot phenol and was separated on a 17% (w/v) 7 M urea denaturing polyacrylamide gel in a 0.5× Tris-borate-EDTA (TBE) electrophoresis buffer. RNA fragments with the length of approximate 20–30 nts or 50–80 nts were extracted by gel crushing and further incubated with an RNA gel extraction buffer (300 mM NaOAc pH 5.2, 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0) overnight.

To control for technical bias during library preparation, appendant mRNA-seq was performed [17]. Specifically, total RNA was extracted using hot phenol, and 75 μg extracted RNA was applied to mRNA purification using the Dynabeads™ mRNA purification kit (Life Technologies, 61006). For fragmentation, 11.1 μL 10× fragmentation reagent (Thermo Fisher Scientific, AM8740) and 100 μL ddH₂O were added to the purified mRNA. Fragmentation proceeded for 30 min at 70 °C and was aborted when 11.1 μL 10× stop solution was added. Fragmented RNA was precipitated by isopropanol overnight and was separated on a 17% (w/v) 7 M urea denaturing polyacrylamide gel. RNA fragments with the length ~ 28 nts were extracted from gel.

The extracted RNA fragments for monosome-seq, disomes-seq, or mRNA-seq were subjected to small RNA library construction for Illumina sequencing (Gnomegen, k02420). The 5′-RNA adaptor contained a 3-nt random sequence at the 3′-end to avoid potential ligation bias. Monosome-seq/RNA-seq and disome-seq libraries were sequenced with single-end 50 and paired-end 100 modes on BGISEQ-500 (BGI Group), respectively.