A549 cells were harvested 5min after treatment with 100 μg/ml cycloheximide (CHX) in DMEM, washed twice in PBS with 100 μg/ml CHX. Samples were lysed with 1 ml mammalian lysis buffer containing 200 µl 5x Mammalian Polysome Buffer, 100 µl 10% Triton X-100, 10 µl 100 mM DTT, 12.5 µl Turbo DNase, 1 µl 100 mg/ml CHX, and 675.5 µl nuclease-free water. After incubation for 20min on ice, lysates were centrifuged at 12,000rpm, 4°C for 10min. For each sample, lysate was divided into two aliquots. For the 600-μl aliquots lysates, 6 units of ARTseq Nuclease was added to each A260 lysate, and the mixtures were incubated for 60min at room temperature with rotation. Nuclease digestion was stopped by 10μL SUPERase·In RNase Inhibitor. Lysates were applied to MicroSpin S-400 HR spin columns. Total RNA was purified with Zymo RNA Clean & Concentrator-25 kit. The rRNA was depleted with Ribo-Zero Gold rRNA Removal Kit. The rRNA-depleted samples were run on a 10% TBE-Urea polyacrylamide gel. RPF between 28-nt and 30-nt were selected. Ribo-seq libraries were then constructed following a protocol described previously. For the 150-μl aliquots lysates, total RNA was isolated with Zymo RNA Clean & Concentrator-25 kit, RNA-seq libraries were constructed with VAHTS Total RNA-seq (H/M/R) Library Prep Kit according to the manufacturer's instructions. All libraries were sequenced with PE150 mode by Illumina Hiseq X10 or NovaSeq 6000.