library_construction_protocol: HEK293T cell line was obtained from ATCC and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were washed twice with 10 mL of ice-cold PBS and placed on ice. Lysis was carried out on plates using 400 μl volume (Tris HCl 20mM pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg/mL Cycloheximide, 1% Triton-X). 5 μl of RNaseI (Invitrogen AM2249) was added to the lysates, followed by a 1 hr digestion at 4°C. Immediately following digestion, ribonucleoside vanadyl complex (NEB S1402S) (20 mM) was added to inhibit further digestion. Digested lysates (400 μl each) were loaded on a sucrose cushion (20mM TrisCl titrated to pH 7.4, 150mM NaCl, 5mM MgCl2, 1% TritonX-100, 34% sucrose, 1 mM DTT, 100 ug/ml cycloheximide) and centrifuged in a SW 41 Ti rotor (Beckman Coulter) at 38,000 rpm at 4°C for 2.5 hours to pellet ribosomes. 700 μl QIAzol Lysis Reagent (Qiagen) was used to resuspend the ribosome pellet, which was was transferred to a fresh 1.5 ml tube and frozen until further processing. To extract RNA, samples were thawed at room temperature and processed with the Qiagen miRNeasy Kit per manufacturer's instructions. RNA was eluted in 20 μl nuclease-free water and size-selected by electrophoresis of 3 μg inputs in a Novex denaturing 15% polyacrylamide TBE-Urea gel. The 26-34 nt RNA fragments were excised, crushed with a pestle, and extracted in 310 μl gel extraction buffer (300 mM NaOAc pH 5.2, 1 mM EDTA, 0.25% w/v SDS) by incubating on dry ice for 30 minutes then overnight at room temperature. The gel-liquid mixture was passed through a Spin-X filter (Corning 8160) and the elution was precipitated with 1.5 μl Glycoblue (5 mM MgCl2, 75% ethanol). RNA was pelleted by centrifugation at 23,300 g at 4°C for 1 hour and resuspended in 10 μl nuclease-free water. For ribosome profiling: 25 ng inputs of size-selected ribosome footprints were immediately processed for library preparation using the D-Plex Small RNA-Seq Kit (Diagenode) per manufacturer's instructions, with some modifications. Approximately half of the resulting cDNA (14 μl) was amplified for 9 cycles. Following quantitation of the target libraries with Agilent HS DNA Kit and Bioanalyzer, the resulting libraries were pooled to equimolar concentrations and cleaned with AMPure 19 XP beads at 1.8x concentration (Beckman Coulter) and eluted in 30 μl TE. To enrich for ribosome footprints in the libraries, the entire elution was size selected in a 3% agarose precast gel (Sage Science) in the BluePippin system using a 172-206 nt range with tight settings. The resulting libraries were sequenced with NovaSeq SP SE 100 (Illumina). For RNA Sequencing: To extract RNA, samples were thawed at room temperature and processed with the Qiagen miRNeasy Kit per manufacturer's instructions. RNA was eluted in 25 μl nuclease-free water and processed with the CATS RNA-seq Kit v2 per manufacturer's instructions (Diagenode C05010045). Approximately half of the resulting cDNA (14 μl) was amplified for 11 cycles. PCR products were cleaned as outlined in the Diagenode protocol and the resulting libraries were sequenced with NovaSeq SP SE 100 (Illumina).