GSM9688392: LPS853 shARG (treatment) Input RNA-Seq -Rep1; Homo sapiens; RNA-Seq
Cancer cells were grown to 80%–90% confluence in DMEM supplemented with 10% FBS in 15-cm dishes. Ribosome footprinting was performed according to TruSeq® Ribo Profile system (Illumina) with modifications. Briefly, cells were treated with 0.1 mg/mL cycloheximide (CHX) for 1 minute to inhibit translation elongation and washed with ice-cold PBS containing 0.1 mg/mL CHX. Next, cells were lysed in 800 μl 1X Mammalian Polysome Buffer (Illumina) supplemented with 1% Triton X-100, 1 mM DTT, 10 units DNase I, 0.1 mg/mL CHX, and 0.1% NP-40. Lysates were cleared by centrifugation at 12,000 g for 10 min at 4°C, and supernatants were flash-frozen in liquid nitrogen and stored at −80°C until processing. RNA concentration of the lysates was measured according to their absorbance at A260 and an equivalent of 12xA260/ml was treated with 5 U/A260 TruSeq Ribo Profile RNase Nuclease (Illumina) for 45 minutes at room temperature. RNase activity was inhibited by adding 15 μl SUPERase to the mixture. Ribosome protected fragments (RPFs) were isolated using MicroSpin S-400 columns (GE Healthcare). RPF RNA samples (5μg) were subjected to ribosomal RNA depletion using RiboMinus Eukaryote Kit v2 (Thermo Fisher Scientific). Ribo-depleted RNA samples were separated on 15% polyacrylamide/TBE/Urea gels (Thermo Fisher Scientific) and the RNA fragments corresponding to ∼25-35 nt were excised. RNA was gel-extracted and precipitated overnight at 4°C using 0.5 M ammonium acetate. In parallel, total RNA input samples were isolated, and fragmented at 94°C for 25 minutes. Input total RNA and RPFs were subjected to end repair by TruSeq Ribo Profile PNK (Illumina), cleaned using RNA Clean & Concentrator-5 kit (ZYMO Research) and ligated to 2.5 μM Universal miRNA Cloning Linker (NEB) by using 100 units T4 RNA Ligase 2, truncated KQ (NEB) for 3 hours at 22°C. After ligation, RNA samples from both total RNA and RPFs were reverse transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) and 0.25 μM RT primer (IDT). cDNA samples were then gel-extracted on 10% polyacrylamide/TBE/Urea gels (Thermo Fisher Scientific) and circularized by CircLigase ssDNA Ligase (Lucigen) for 2 hours at 60°C. cDNA libraries for total RNA and RPFs were amplified for 9 and 12 PCR cycles, respectively, using Phusion High-Fidelity PCR Master Mix (NEB), Illumina index primers and 10 μM forward primer (IDT). Amplified libraries were cleaned using AMPure XP Beads (Beckman Coulter), followed by gel-extraction on 8% native TBE gels (Thermo Fisher Scientific). Libraries were sequenced with Illumina NextSeq 500.