GSM9237614: siNC repeat 3; Homo sapiens; RNA-Seq
For RNA extraction, the Trizol method is utilized. Cells are first washed twice with PBS. 1ml of Trizol reagent is added to the cell culture dish, and cells are thoroughly dissolved by repetitive pipetting or scraping. The Trizol-cell mixture is transferred to an EP tube, to which 200μL of chloroform is added and mixed thoroughly. It is then centrifuged at 4°C for 10 minutes at 12000g. The supernatant is carefully transferred to a new RNase-free centrifuge tube, and an equal volume of RNase-free alcohol is added and mixed. The RNA is then precipitated in a -20°C freezer. After another round of centrifugation, the supernatant is discarded, and the residue is washed with 70% ethanol. Finally, the precipitate is dissolved in DEPC water to obtain the final RNA, which is stored at -80°C. Total RNA was extracted from samples using standard protocols. For transcriptomic library construction, the VAHTS Universal V5 RNA-seq Library Prep Kit (Vazyme) was employed according to the manufacturer's instructions. This kit facilitates high-efficiency and high-fidelity library preparation, which is critical for accurate transcriptome profiling. The resulting libraries were assessed for quality and quantity using an Agilent Bioanalyzer 2100 system and Qubit fluorometer. Libraries passing quality control were sequenced on the Illumina NovaSeq 6000 platform to generate 150 bp paired-end reads, enabling robust transcript coverage and reliable downstream analysis.