Sucrose solutions were prepared in polysome extraction buffer (10 mM HEPES, 100 mM KCl, 5 mM MgCl2, pH 7.4, 100 ug/ml Cycloheximide, 2 mM DTT). Sucrose gradients were prepared in SW41 ultracentrifuge tubes by mixing 15% and 45% sucrose solution using a BioComp Gradient Master 108. 20M cells per sample per condition were resuspended after 3 hours of culture in nutrient stress media, washed once with PBS containing 50ug/mL cycloheximide, and lysed in 500uL polysome extraction buffer containing 1% Triton-X and SUPERAse In (Thermo Scientific, #AM2696), followed by 30min incubation on ice with vortexing at 5min intervals. Lysates were centrifuged for at 12000g for 15 min at 4C, then 50uL lysate was snap frozen as “Input” RNA and 400uL lysate was loaded onto 15–45% sucrose gradients followed by centrifugation at 37K rpm for 2hr at 4°C in a SW41 rotor of an Optima XPN 80 Beckman ultracentrifuge. Gradients were fractionated with a speed of 800uL/min using a Biocomp piston gradient fractionator, which recorded the OD254nm. Fractions corresponding to 60 sec intervals were collected, labeled 1-15, and stored at -80c before RNA extraction. 250uL of each sucrose fraction was aliquoted for RNA isolation. 20uL of input RNA was diluted to 250uL with molecular biology water before processing. 750uL of Trizol LS containing 20pg of luciferase control RNA (Promega, #L4561) was added to each sample and thoroughly mixed. 1mL of 100% ethanol was added to each sample and thoroughly mixed. Each sample was loaded into a separate column of a Zymo Direct-Zol-96 plate and purified according to manufacturer's instructions. Eluted RNA was quantified on a Nanodrop, and a pooled sample containing an equal volume of the RNA from polysome-bound fractions (fractions 9-13) was generated for downstream sequencing. For all Control, NoArginine, NoBCAA, NoGlucose, NoGlutamine, and NoMethionine samples: RNA samples were checked for quality using an Agilent tapestation and libraries were prepared using the Illumina TruSeq Stranded Total RNA Gold according to manufacturer's instructions. For all DMSO, Torin, 4EGi1, and Halo samples: Samples were checked for quality using a tapestation. Messenger RNA was purified from total RNA using poly-oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers. Then the second strand cDNA was synthesized using dUTP, instead of dTTP. The directional library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. After library quality control, different libraries were pooled based on effective concentration and targeted data amount.