RNA-Seq of hepatocellular carcinoma: HepG2 cells, test
HepG2 cells were seeded into 6well plates at 1.5 105 cells per well and treated with 100 M of SCU for 48 h at 37C of 5% CO2. After incubation, total RNAs were extracted using TRIzol (Ambion, Thermo Scientific, Rockford, IL, USA). The concentration of RNA was determined using a spectrophotometer (BioTek, Winooski, VT, USA). Isolated total RNA was then subjected to sequencing to obtain expression data. The mRNA sequencing was prepared by Theragenbio (Seongnam-si, Gyeonggi-do) using the following protocol (Table 1). The libraries were prepared for 151bp paired-end sequencing using TruSeq stranded mRNA Sample Preparation Kit (Illumina, CA, USA). Utilizing oligo (dT) magnetic beads, mRNA molecules were specifically isolated and fragmented from 1 g of total RNA. The fragmented mRNAs were synthesized as single-stranded cDNAs through random hexamer priming. Double-stranded cDNA was created by using this as a template for second-strand synthesis. After the sequential process of end repair, A-tailing, and adapter ligation, cDNA libraries were amplified with PCR (Polymerase Chain Reaction). The quality of these cDNA libraries was evaluated with the Agilent 2100 BioAnalyzer (Agilent, CA, USA). According to the manufacturer's library quantification methodology, they were measured using the KAPA library quantification kit (Kapa Biosystems, MA, USA). Following cluster amplification of denatured templates, sequencing was progressed as paired-end (2 151bp) using Illumina NovaSeq6000 (Illumina, CA, USA).