GSM8634584: HCCLM3 cells, quercetin 50μM, rep1; Homo sapiens; RNA-Seq
Total RNA was extracted from the tissues using Trizol (Invitrogen, Carlsbad, CA, USA) according to manual instruction. About 60 mg of tissues were ground into powder by liquid nitrogen in a 2 mL tube, followed by being homogenized for 2 minutes and rested horizontally for 5 minutes. The mix was centrifuged for 5 minutes at 12,000×g at 4°C, then the supernatant was transferred into a new EP tube with 0.3 mL chloroform/isoamyl alcohol (24:1). The mix was shacked vigorously for 15s, and then centrifuged at 12,000×g for 10 minutes at 4°C. After centrifugation, the upper aqueous phase where RNA remained was transferred into a new tube with equal volume of supernatant of isopropyl alcohol, then centrifuged at 13,600 rpm for 20 minutes at 4°C. After deserting the supernatant, the RNA pellet was washed twice with 1 mL 75% ethanol, then the mix was centrifuged at 13,600 rpm for 3 minutes at 4°C to collect residual ethanol, followed by the pellet air dry for 5-10 minutes in the biosafety cabinet. Finally, 25µL~100µL of DEPC-treated water was added to dissolve the RNA. Subsequently, total RNA was qualified and quantified using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA) Oligo(dT)-attached magnetic beads were used to purified mRNA. Purified mRNA was fragmented into small pieces with fragment buffer at appropriate temperature. (Note: for FFPE samples, the above two steps can be omitted according to the quality of samples.) Then, First-strand cDNA was generated in First Strand reaction system by PCR, and the second-strand cDNA was generated as well. The reaction product was purified by magnetic beads, afterwards, A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments with adapters were amplified by PCR, and the products were purified by Ampure XP Beads. The library was assessed quality and quantity using the Agilent 2100 bioanalyzer. Then, undergo DSN treatment. The DSN treated library was assessed quality to ensure the high quality of the sequencing data by two methods: checked the distribution of the fragments size using the Agilent 2100 bioanalyzer, and quantified the library using real-time quantitative PCR (qPCR). The qualified library was amplified on cBot to generate the cluster on the flowcell. And the amplified flowcell was sequenced single end on the BGI-500 platform (BGI-Shenzhen, China)