Zymo column

Total RNA was extracted from cells with Triol (Ambion, cat#15596018) and DNase treated, cleaned and concentrated using Zymo columns (Zymo Research, cat#R1013) following manufacturer’s instructions. RNA integrity number (RIN) was measured using BioAnalyzer (Agilent Technologies) RNA 6000 Nano Kit to confirm RIN above 7 for each sample. The cDNA libraries, next generation sequencing, and bioinformatics analysis was performed by Novogene.

Eight culture plates per condition (~23 million cells per condition) were incubated with harring-tonine (2μg/mL) for 2 minutes at 37°C followed by 5 minutes of cycloheximide (100μg/mL) treatment at 37°C. Cells were washed twice with PBS after each treatment. Cells were scraped in 600uL of lysis buffer (50mM HEPES, 75mM KCl, 5mM MgCl2, 250mM sucrose, 0.1mg/mL cycloheximide, 2mM DTT, 1% Triton X-100 and 1.3% sodium deoxycholate and 5μl of RNase OUT) on ice. Lysates were rocked for 10 minutes at 4°C and centrifuged at 3000g for 15 minutes at 4°C. 400μl of lysates supernatant (cytosolic cell extracts) were layered over cold sucrose gradients (10mM HEPES, 75mM KCl, 5mM MgCl2, 0.5mM EDTA and increasing sucrose concentrations from 20% to 47%). Gradients were centrifuged at 34,000 rpms in a Beckman SW41 rotor for 2h and 40 minutes at 4°C. After centrifugation, low (0 to 2 ribosomes) and high (>2 ribosomes) polysome fractions were collected in Trizol (1:1) using a density gradient fractionation system (Brandel) equipped with a UA-6 absorbance detector and a R1 fraction collector. RNA was DNase treated, cleaned, and concentrated using Zymo columns (Zymo Research, Cat# R1013). The cDNA libraries, next generation sequencing, and bioinformatics analysis was performed by Novogene.

Total RNA was extracted from cells with Trizol, Dnase-treated, cleaned, and concentrated using Zymo columns (Cat# R1013; Zymo Research) following the manufacturer’s instructions. Optical density values of RNA were measured using NanoDrop One (Thermo Fisher Scientific) to confirm an A260 and A280 ratio above 1.9. Relative expression of target genes (Table S3) was analyzed using the QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) with clear 96-well plates (Greiner Bio-One). Primers were designed using the Integrated DNA Technologies (IDT) tool (http://eu.idtdna.com/scitools/Applications/RealTimePCR/). A total of 25 ng of RNA was used for One-Step qPCR (Quanta BioSciences) following the manufacturer’s instructions in a final volume of 10 μl. Conditions for amplification were 10 min at 48°C, 5 min at 95°C, 40 cycles of 10 s at 95°C, and 7 s at the corresponding annealing temperature (Table S3). The assay ended with a melting curve program: 15 s at 95°C, 1 min at 70°C, and then ramping to 95°C while continuously monitoring fluorescence. Each sample was assessed in triplicate. Relative quantification was determined to multiple reference genes (B2M, MRPL9, PSMC4, and PUM1) using the delta–delta Ct method.