GSM4879485: OSCC SAS siD mRNA-seq; Homo sapiens; RNA-Seq
For RNAseq, total RNA was extracted using Trizol Reagent (ThermoFisher) according to the manufacturer's instructions. Ribosomal RNA (rRNA) was depleted using the Ribo-off rRNA Depletion Kit (Vazyme). For Riboseq, cells were lysed with the polysome buffer containing 20 mM Tris-HCl pH7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 200 µg/ml cycloheximide, 1% (v/v) Triton X-100 and 25 U/ml Turbo DNase I (Ambion). The lysates were subsequnetly digested with RNase I (Ambion). After purification, rRNA was depleted using the Ribo-off rRNA Depetion Kit (Vazyme). Ribosome-protected fragments (RPF) were purified with PAGE and dephosphorylated with T4 Polynucleotide Kinase (New England BioLabs). For RNAseq, library preparation was performed using the NETNext Ultra Directional RNA Library Prep Kit for Illumina (New England BioLabs) according to the manufacturer's instructions. For RIBOseq, linkers were ligated to RPFs using T4 RNA ligase 2, truncated K227Q (New England BioLabs) and the cDNA was synthesized using SuperScript III reverse transcriptase (ThermoFisher). The cDNA samples were circularized by CircLigase ssDNA Ligase (Lucigen), amplified by Phusion High-Fidelity DNA Polymerase (New England BioLabs) and purified using AMPure XP for PCR Purification (Beckman Coulter). The quality and quantity of the libraries were respectively analyzed by Fragment Analyzer Automated CE System (Advanced Analytical Technologies) and Qubit Fluorometer (ThermoFisher).