Each 5,000 U total ribosomes were digested by 1,500 U RNase I (Ambion, AM2294) for RF generation. The digested solutions were separated by profiling as described in the polysome profiling experiments. We added 2 volume 8 M guanidine hydrochloride, 3 volume ethanol, and 2 µL GlycoBlue (Ambion, AM9516, 15 mg/mL) into monosome fractions for RNA extraction. The ~28-nt RFs were separated with Urea-PAGE, and rRNA was removed using DNA probes complementary to rRNA sequences. Then, RNase H and DNase I were used to digest the probes. RFs were purified using magnet beads (Vazyme). After obtaining RFs above, Ribo-seq libraries were constructed using NEBNext Multiple Small RNA Library Prep Set for Illumina (catalog no.E7300S, E7300L). Briefly, adapters were added to both ends of RFs, followed by reverse transcription and PCR amplification. The 140 to 160-bp size PCR products were enriched to generate a cDNA library and sequenced using Illumina HiSeqTM 2500 by Gene Denovo Biotechnology Co.
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