library_construction_protocol: On the day of collection, 5%-50% sucrose gradient was prepared in ultra-centrifuge tolerant tube by combining 5% sucrose buffer (20 mM HEPES (pH 7.6), 100 mM KCl, 5 mM MgCl2, 5% sucrose, 100 µg/ml CHX, 1% protease inhibitor, 1% RNase inhibitor) with 50% sucrose buffer (20 mM HEPES (pH 7.6), 100 mM KCl, 5 mM MgCl2, 50% sucrose, 100 µg/ml CHX, 1% protease inhibitor, 1% RNase inhibitor)  using Gradient Station (Biocomp). The sucrose gradient for all samples was stored in cold room (4 ºC) before use. Cells were then treated with 100 µg/ml cycloheximide (CHX) in complete medium for 7 min at 37 ºC followed by immediate wash with ice-cold PBS buffer containing 100 µg/ml CHX twice. The cells were collected in 5 ml ice-cold PBS buffer containing 100 µg/ml CHX and pelleted by centrifugation at 500× g for 5 min. Cells from 4× 15-cm plates were combined as one sample. 4× volumes of lysis buffer (20 mM HEPES (pH 7.6), 100 mM KCl, 5 mM MgCl2, 1% Triton X-100, 100 µg/ml CHX, 1% protease inhibitor, 1% RNase inhibitor) was added to the combined cell pellet and the cells were lysed on ice for 20 min with periodic perturbation (or rotating at 4 ºC). Supernatant lysate of each sample was collected after centrifugation at 16,000× g for 15 min. 4 µl Turbo DNase was added to the lysate and incubated at room temperature for 15 min. The lysate samples were centrifuged at 16,000× g for 15 min after DNase treatment to remove any debris. OD260 of each lysate sample was measured using Nanodrop. The OD260 values of all samples were adjusted to the same using lysis buffer. 20 µl of the lysate was saved for Western blot validation. 1/5 of the lysate was saved as the input and the total RNA was extracted using TRIzol reagent. All the sucrose gradient tubes were weighed and balanced and 500 µl gradient solution was slowly removed from the top before loading. Equal amount (~500 µl) of the 4/5 lysate were slowly and horizontally added to the top of the gradient while gently rotating the tube. The gradient tubes were then centrifuged at 28,000 rpm and 4 ºC under vacuum for 3 hours using ultracentrifuge (Beckman Coulter Optima L-100XP, SW28.1 rotor). The sucrose gradient fractions (30 fractions, 590 µl each) were collected automatically using Sucrose Gradient and fraction collector (Gilson). All fractions were flash frozen using liquid nitrogen before RNA extraction and Western blot validation. 400 µl aliquot from disome and above (polysome) fractions were used to extract total RNA. Total RNA from these fractions were combined for polysome associated tRNA sequencing and polysome mRNA extraction and sequencing. Polysome mRNA sequencing libraries were constructed using MSR-seq. Briefly, 400ng input and polysome polyA+ RNA was fragmented at 94 ºC for 3 min using Magnesium fragmentation buffer (NEB, E6150S) to get ~300 nt fragment RNA. Fragment RNA larger than 200 nt was then purified using RNA clean and concentrator kit and eluted in 8 µl sterile H2O. 1 μl T4 PNK buffer and 1 µl 10 U/μl T4 PNK (NEB M0201L) were added to the fragment RNA and incubated at 37°C for 30 min. T4 PNK was inactivated by incubation at 75 °C for 10 minutes followed by immediately incubation on ice. The entire reaction mixture (~10 μl) was used as the RNA input for MSR-seq.