Original sequencing data
4sU Labeling of Cells
HEK293T cells were labeled with 4-thiouridine (4sU) for 30 min, 1 h, or 2 h. All 4sU-containing solutions were protected from light throughout the procedure. Cells were seeded at a density of 5 × 10⁶ cells per dish the day before the labeling experiment. For pulse labeling, 500 μM 4sU was added to achieve the final concentration.
Polysome Profiling
To prepare DEPC-treated water, 1 mL of DEPC was added to 1 L of deionized water, mixed thoroughly, and incubated at room temperature for 12 h before autoclaving. For the 60% (w/v) sucrose solution, 60 g of sucrose was dissolved in deionized water to a final volume of 100 mL. To eliminate any residual RNase, 100 μL of DEPC was added to the solution, which was then incubated at room temperature for 12 h and subsequently autoclaved.
All necessary buffers were freshly prepared. The 10× salt solution consisted of 200 mM HEPES-KOH (pH 7.5), 1 M KCl, 50 mM MgCl₂, and DEPC-treated water. The lysis buffer was prepared by combining 1× salt solution, 2% Triton X-100, a protease inhibitor cocktail (1×), 200 μg/mL cycloheximide (CHX), 1 mM DTT, and 40 U/mL RNase inhibitor (Promega N2515). For the sucrose gradients, a 10% sucrose gradient buffer was prepared by adding 6.64 mL of the 60% sucrose solution and 4 mL of the 10× salt solution to DEPC-treated water, along with protease inhibitors (1×), 200 μg/mL CHX, 1 mM DTT, and 4 U/mL RNase inhibitor, to a final volume of 40 mL. Similarly, the 45% sucrose gradient buffer was prepared by adding 30 mL of the 60% sucrose solution and 4 mL of the 10× salt solution, with the same additives, to a final volume of 40 mL.
Before polysome profiling, cells were incubated with 200 μg/mL CHX for 10 min at 37°C to inhibit translation. Cells were then placed on ice, and the medium was aspirated. The cells were washed with ice-cold DPBS containing 200 μM CHX, followed by the addition of 600 μL of lysis buffer directly to the dish. Cells were scraped, collected, and lysates were centrifuged at 20,000 g for 10 min at 4°C. For HEK293T or suspension cells, the DPBS washing step was omitted; cells were harvested by pipetting and centrifuged at 300 g for 3 min at 4°C.
Sucrose gradients were prepared using a Biocomp Gradient Master programmed for a 10%–45% gradient suitable for the SW41Ti rotor. Each sample (400 μL) was carefully loaded onto the gradient, and centrifugation was performed at 36,000 rpm for 1.5 h at 4°C using the SW41Ti rotor in a Beckman Optima L8-80XP ultracentrifuge. After centrifugation, the gradient was fractionated into 12 fractions. Fractions corresponding to free RNA, ribosomal subunits (40S, 60S), monosomes (80S), and polysomes were collected and combined into four pooled fractions representing different ribosome density levels. Samples were immediately frozen at −80°C and stored for no longer than one week.
RNA Purification
For RNA extraction from polysome fractions, 1 mL of each sample was supplemented with either 1 µg of yeast total mRNA as an endogenous spike-in or 1 ng of IVT mRNA as an IVT library spike-in. RNA was extracted by adding 750 μL of TRIzol reagent, vortexing for 15 s, and incubating at room temperature for 5 min. Next, 200 μL of chloroform was added, and samples were vortexed for 15 s, followed by a 2–3 min incubation at room temperature. Samples were centrifuged at 16,000 × g for 15 min at 4°C, and the aqueous phase containing RNA was carefully transferred to a new tube. RNA was precipitated by adding 1 μL of glycogen (20 mg/mL) and an equal volume of isopropanol, followed by centrifugation at 16,000 × g for 30 min at 4°C. The RNA pellet was washed with 1 mL of 75% ethanol, centrifuged at 16,000 × g for 5 min at 4°C, air-dried for 3–5 min, and resuspended in 20–50 μL of RNase-free water. RNA concentrations were determined, and samples were stored at −80°C.
Library Preparation
To detect 4sU-labeled RNA, thiol modification was performed using iodoacetamide (IAA). The reaction mixture was prepared by combining 0.5–20 μg of RNA, 5 μL of DPBS, 5 μL of IAA (100 mM), 25 μL of DMSO, and RNase-free water to a final volume of 50 μL. The mixture was incubated at 50°C for 15 min. The reaction was stopped by adding 1 μL of 1 M DTT.
To precipitate the RNA, 1 μL of glycogen (20 mg/mL), 5 μL of sodium acetate (3 M, pH 5.2), and 125 μL of 100% ethanol were added to each sample, followed by vortexing. The samples were incubated at −80°C for 30 min, then centrifuged at 16,000 × g for 30 min at 4°C. The RNA pellet was washed twice with 1 mL of 75% ethanol and centrifuged again at 16,000 × g for 10 min at 4°C. The supernatant was removed, and the pellet was air-dried for 3–5 min. The RNA was resuspended in 5–10 μL of nuclease-free water. RNA was stored at −80°C.
RNA purified after thiol modification was first subjected to mRNA enrichment using mRNA beads (Vazyme). To generate full-length cDNA, the purified mRNA was mixed with 1 μL of poly-dTVN primer (10 μM) and nuclease-free water to a final volume of 5 μL. This mixture was heated to 70°C for 5 min to denature secondary structures and immediately chilled on ice. Reverse transcription was performed by adding 1 μL of 10× M-MuLV RT buffer (NEB), 1 μL of TSO (1 μM), 0.5 μL of M-MuLV reverse transcriptase (20 U/μL), 0.5 μL of dNTPs (10 mM), 0.1 μL of RNasin (40 U/μL), and 1.9 μL of nuclease-free water. The reaction was incubated at 42°C for 90 min, followed by heat inactivation at 85°C for 5 min, and held at 4°C.
For cDNA amplification, 10 μL of the reverse transcription mix was combined with 5 μL of 5× KAPA HiFi Buffer (Roche), 0.5 μL of KAPA HiFi HotStart polymerase, 0.25 μL of ISPCR primer (10 μM), 0.75 μL of dNTPs (10 mM), and 8.5 μL of nuclease-free water. PCR was performed using the following cycling conditions: an initial denaturation at 98 °C for 45 s, followed by 10 cycles of 98°C for 10 s, 67°C for 15 s, and 72°C for 3 min, with a final extension at 72°C for 5 min. The PCR product was purified using VAHTS DNA Clean Beads (Vazyme) according to the manufacturer's instructions.
Tn5 transposase was preloaded with Tn5ME-A and Tn5ME-B linkers as previously described by Hennig et al1. For tagmentation, the reaction was prepared by mixing 2.5 μL of freshly prepared tagmentation buffer (40 mM Tris-HCl pH 7.5, 40 mM MgCl₂), 1.25 μL of purified cDNA (1–50 ng/μL), and 1.25 μL of Tn5 working solution. The mixture was incubated at 55°C for 10 min, followed by the addition of 1.25 μL of 0.2% SDS and further incubation at room temperature for 5 min.
The tagmented cDNA was amplified by PCR. The reaction mixture included 5 μL of the tagmentation product, 1.2 μL of 5× KAPA Buffer, 0.75 μL of 100% DMSO, 1.25 μL each of i5 and i7 adapter index primers (10 μM), 0.45 μL of dNTPs (10 mM), 0.3 μL of KAPA HiFi HotStart polymerase, and 4.8 μL of nuclease-free water, for a final volume of 15 μL. The thermocycler program was as follows: gap filling at 72°C for 3 min; initial denaturation at 95°C for 30 s; 12 cycles of 98°C for 20 s, 58°C for 15 s, and 72°C for 30 s; and a final extension at 72°C for 3 min.
The amplified product was visualized using a 3% agarose gel. DNA fragments between 250 bp and 400 bp were excised from the gel. DNA was purified using the Magen Gel Extraction Kit (Magen Biotechnology) according to the manufacturer's protocol. The purified library was quantified using Qubit and sequenced on a NovaSeq platform with 150 bp paired-end reads.
Inhibitor Treatment in HEK293T Cells
To assess the effect of m6A depletion on newly transcribed mRNA, HEK293T cells were treated with STM2457 at a final concentration of 20 µM (MCE: HY-134836) during 4sU labeling for 2 hours. This treatment inhibits METTL3, reducing m6A modification on nascent transcripts.
For translation inhibition, puromycin (Gibco: A1113803) was added to the culture medium at a final concentration of 1 µg/mL during 4sU labeling for 2 hours.
After treatment, cells were processed using the previously described method for polysome profiling and other downstream analyses.
THP-1 Cell Differentiation and Activation
THP-1 cells were cultured to the appropriate density for differentiation. Phorbol 12-myristate 13-acetate (PMA) was added to the culture medium at a final concentration of 5 ng/mL to induce differentiation. The cells were incubated in PMA-containing medium for 24 h, during which they adhered to the culture dish and began to exhibit macrophage-like morphology. After 24 h, the medium was replaced with fresh, PMA-free medium, and the cells were incubated for an additional 72 h to allow for resting and stable differentiation into macrophages. These differentiated and rested THP-1 macrophages were then used for subsequent activation experiments or other functional assays. For activation, lipopolysaccharide (LPS) was added to the rested THP-1 macrophages at a final concentration of 1 μg/mL.
Proteomics for HEK293T Cells and THP-1 Macrophages
A total of 1 × 10⁶ cells were collected and centrifuged at 300 × g for 3 min, and the supernatant was discarded. This washing step was repeated three times by resuspending the cells in DPBS and centrifuging to remove serum proteins and trypsin. After the final wash, the cells were resuspended in DPBS, and the protein concentration of the cell suspension was quantified using the BCA method.
Next, an aliquot containing 20 μg of protein was centrifuged at 300 × g for 3 min and the supernatant was discarded. The pellet was resuspended in 15 μL of DPBS and 5 μL of 5× SDS loading buffer, then mixed by vortexing. The sample was heated at 100°C for 10 min using a thermocycler and immediately placed on ice afterward. A 10% SDS-PAGE gel was prepared, and 20 μL of the sample was loaded onto the gel. The gel was run for 30 min. Afterward, all bands were excised from the gel, and in-gel digestion with trypsin was performed. The resulting peptides were analyzed using an Orbitrap Fusion Lumos mass spectrometer, and protein abundances were determined based on the mass spectrometry data.
