Cells were lysed directly on plates on ice in 500 μL ice-cold polysome lysis buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 1% Triton X-100, freshly supplemented with 20 U/mL SUPERase-In RNase inhibitor, 24 U/mL TURBO DNase, 1 mM DTT, 100 μg/mL cycloheximide, and 1× EDTA-free protease inhibitor cocktail. Lysates were clarified by centrifugation at 20,000 × g for 2 min at 4°C and were either flash-frozen or used immediately for monosome fractionation. For monosome fractionation, lysates were supplemented with 5 mM CaCl₂ and digested with micrococcal nuclease at 3 U/μg RNA for 30 min at room temperature. Digestion was quenched by addition of EGTA to a final concentration of 6.25 mM, and samples were loaded onto sucrose gradients as previously described in Jan, Williams, and Weissman (2014). Gradients were centrifuged for 2 h at 41,000 rpm in an SW-41 rotor, and monosome fractions were collected using a BioComp gradient fractionation system coupled to a Gilson FC203B fraction collector and Triax software. RNA was extracted from collected monosome fractions using TRIzol reagent according to the manufacturer's instructions. Ribosome-protected fragment libraries were prepared using barcoded linkers as previously described in Hacisuleyman et al (2024). Indexed libraries were pooled and sequenced on an Illumina NextSeq 2000 Sequencing System using a P3 flow cell.