library_construction_protocol: Protocol 1 and 2: If cells were adherent, they were detached cells with TrypLE. Cells were spun down for 5 min at 210 g. We resuspended the cells in 1 ml of cold PBS and placed them on ice. We took 25 µl of each sample, added 175 µl of PBS, and measured the GFP fluorescence on the Attune NxT flow cytometer to determine cell numbers and transfection efficiencies. We used the Monarch Total RNA Miniprep Kit (NEB, T2010S) to purify total RNA. We eluted in 100 µl of nuclease-free water and determined concentrations using a NanoDrop 2000c (Thermo Fisher). mRNA was purified using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB, E7490L). We eluted in 18 µl of nuclease-free water. mRNA was reverse transcribed and amplified using custom primers. Protocol 3: Cells were pelleted (100 g RT, then 1000 g 4°C after PBS wash) and frozen at −80°C. The appropriate spike-in amount was calibrated using a t=0 sample by testing serial spike-in dilutions and estimating relative spike-in vs. cellular RNA abundance via qPCR. The selected spike-in amount (targeting the highest spike-in species at ~2x the most abundant cellular mRNA at t=0) was added to all samples during lysis (NEB RNA miniprep kit), followed by library preparation and sequencing as described above. Protocol 4: Cells were treated with cycloheximide and lysed. Media was replaced with 5 mL RT DPBS + CHX, incubated 5 min at 37°C, replaced with ice-cold DPBS + CHX, and cells were lysed in 300 µL cold lysis buffer. Lysates were incubated on ice (10 min), homogenized (25G needle), centrifuged (16,000 g, 5 min, 4°C), treated with DNase I (0.005 U/µL, 30 min, ice), and stored at -80°C. Polysome profiling was performed using 20–55% sucrose gradients (100 mM KCl, 20 mM HEPES pH 7.2, 10 mM MgCl₂) prepared by overnight diffusion at 4°C. Lysates were layered onto gradients and ultracentrifuged (151,000 g, 3 h, SW-41 Ti rotor). ~25 fractions were collected; free RNA and visible polysome peak fractions were retained for downstream analysis. Spike-in calibration was performed on total lysate by testing serial spike-in dilutions followed by Trizol extraction and RNA purification (Zymo RNA C&C-5 kit). qPCR curves were used to determine the spike-in amount targeting the most abundant spike-in species at ~2x the most abundant endogenous mRNA in the average fraction. All fractions were then processed with spike-in addition, Trizol extraction (scaled volumes), column purification, and standard library preparation. Fractions corresponding to the same polysome peak were combined during purification. mRNA isolation (magnetic beads) was performed for total lysate and free ribosome fractions only; ribosome-bound fractions were assumed to be mRNA-enriched. Custom workflow