Automated fractionation system

Sucrose solutions were prepared in polysome buffer (10 mM HEPES, pH 7.4, 100 mM KCl, 5 mM MgCl2 and 100 mg/ml cycloheximide). A 15–45% (w/v) sucrose density gradient was freshly prepared in a SW41 ultracentrifuge tube (Backman) using a Gradient Master (BioComp Instruments). Cells were lysed in polysome lysis buffer (polysome buffer and 2% TritonX-100) and cell debris were removed by centrifugation at 13,000 × g for 10 min at 4 °C. In all, 500 mL of supernatant was loaded onto sucrose gradients followed by centrifugation for 2 h 30 min at 36,000 × g and 4 °C in a SW41 rotor. Separated samples were fractionated at 0.75 ml/min through an automated fractionation system (Isco) that continually monitors OD254 values. An aliquot of each ribosome fraction were spiked with isolated luciferase RNA and then used to extract total RNA using Trizol LS reagent (Invitrogen) for RT-qPCR.

Cells were grown on 150 mm dishes as described above. At 5 min before cell collection, cycloheximide was added to the medium at a concentration of 100 µg/mL. Harvested cells were washed with ice-cold PBS containing cycloheximide (100 µg/mL). Cells were lysed in 600 μL of polysome lysis buffer (10 mM HEPES, pH 7.4, 100 mM KCl, 5 mM MgCl₂) containing cycloheximide (100 µg/mL) and Triton-X100 (1% v/v). The lysate was centrifuged at 12,000 × g for 15 min at 4 °C. An aliquot was removed for western blotting, and 600 µl of supernatant was loaded onto 15–45% w/v sucrose density gradients, which were freshly prepared in SW41 ultracentrifuge tubes, followed by centrifugation at 32,000 rpm for 150 min at 4 °C in a SW41 rotor. Separated samples were collected from the top of the sucrose density at 0.375 mL/min through an automated fractionation system (Isco), and A₂₅₄ values were continuously monitored and recorded. A₂₅₄ profiles were scanned and traced (Inkscape). Polysome/monosome (P/M) ratios of the traced profiles were determined by aligning profiles to the bottom of the monosome peak and measuring the areas below the monosome and polysome peaks to a line equal to the lowest point on each analysis set.

Cells at 80–90% confluence were changed with fresh medium to remove the dead cells 3–4 h before harvesting. Four 10 cm dishes of cells were harvested in 450 µl lysis buffer (10 mM HEPES, pH 7.4, 100 mM KCl, 5 mM MgCl₂, 1% Triton X-100) containing CHX (100 µg ml⁻¹), then centrifuged at 12,000g, 4 °C for 10 min. The supernatant was collected and subjected to sucrose gradient sedimentation. Sucrose solutions were prepared in polysome buffer (10 mM HEPES, pH 7.4, 100 mM KCl, 5 mM MgCl₂). 15–45% (w/v) sucrose density gradients were freshly made in SW41 ultracentrifuge tubes (Backman) using Gradient Master (BioComp Instruments). Totally, 500 µl of the cell lysates was loaded onto sucrose gradients followed by centrifugation for 2.5 h at 32,000 rpm, 4 °C in a SW41 rotor. Separated samples were fractionated at 1.5 ml min⁻¹ through an automated fractionation system (Isco) that continually monitors OD254 values.

For Ribo-seq, five 10 cm dishes of cells were harvested in 450 µl lysis buffer (1% Triton X-100 in polysome buffer) containing CHX (100 µg/ml), then centrifuged at 12,000 g 4 °C for 10 min. The supernatant was collected and subjected to sucrose gradient sedimentation. Sucrose solutions were prepared in polysome buffer. 15%- 45% (w/v) Sucrose density gradients were freshly prepared in a SW41 ultracentrifuge tube (Backman) using a Gradient Master (BioComp Instruments). Supernatant was loaded onto sucrose gradients followed by centrifugation for 2 h 30 min at 32,000 rpm 4 °C in a SW41 rotor. Separated samples were fractionated at 1.5 ml/min through an automated fractionation system (Isco) that continually monitors OD254 values. For both QTI-seq and Ribo-seq, ribosome fractions separated by sucrose gradient sedimentation were pooled and digested with E. coli RNase I (Ambion, 750 U per 100 A260 units) by incubation at 4 °C for 1 h. SUPERase•In (50 U per 100 U RNase I) was then added into the reaction mixture to stop the digestion. Total RNA was extracted using TRIzol LS reagent. Purified RNA was used for cDNA library construction and high-throughput sequencing described below.