Ribosome profiling sequencing (Ribo-seq) in Huh7 control and SMYD5 KO cells upon normal condition or Torin1 treatment

To understand how RPL40 K22me3 transferred by SMYD5 affected ribosome function on normal culture condition or after mTOR inhibition, we conducted ribosome profiling sequencing (Ribo-seq) in Huh7 control (NC) and SMYD5 KO cells. Huh7 control (NC) and SMYD5 KO cells were culturedHuh7 cells were cultured in DMEM supplemented with 10% FBS and 1% P/S or treated by 50 nM Torin1 for 12 hrs before collecting. Ribo-seq was performed as ARTseq Ribosome Profiling Kit’s instructions with modifications. About 10^7 cells were pre-treated by 100 μg/ml cycloheximide (MCE) for 10 min at 37℃, then washed and collected by ice-cold PBS containing 100 μg/ml cycloheximide. Cells were lysed by 120 μl Mammalian Polysome Buffer (10 mM Tris-HCl pH 7.5, 100 mM KCl, 5 mM MgCl2, 1% Trion X-100 with 1x protease inhibitor cocktail (Roche)) for 10 min. After centrifugation at 13,000 g for 10min at 4℃. 10 ul lysate was kept for input and purified by RNA Clean & Concentrator-5 kit (RCC-5 R1016, zymo), followed by rRNA removal with Ribo-off rRNA Depletion (N406, Vazyme) and library preparation with VAHTS Universal RNA-seq Library Prep Kit (NR605, Vazyme). Add about 30U RNase I (EN0601, Thermo) to 100ul lysate and digest for 45 min at room temperature. Digestion was stopped by the addition of 4 μl of Superase-In (AM2696, Thermo). Meanwhile, MicroSpin S-400 HR columns (27514001, Cytiva) were equilibrated with 3 ml of Mammalian Polysome Buffer by gravity flow and emptied by centrifugation at 600g for 4 min. We then immediately loaded 100 μl of the digested lysate on the column and eluted the column by centrifugation at 600g for 2 min. RNA was extracted by RCC-5 and separated on 15% denaturing urea-PAGE gel. After SYBR gold (S11494, Thermo) staining, the size ranges from 25 nt to 40 nt was cut out and recovered by small-RNA PAGE Recovery Kit (R1070, zymo). The eluted RNA were mix with Superase-In, T4 PNK and Buf A (EK0031, Thermo) at 37 ℃ for 15 min and supplemented by 1 mM ATP (R0441 ) for another 30 min before extraction by RCC-5 and generation library by Small RNA Library Prep Kit (NR811, Vazyme). The libraries were sequenced by NovaSeq 6000 (conducted by Nanjing Gaoxin Precision Medicine Technology Co., Ltd).