Illumina NovaSeq 6000 paired end sequencing; YTHDF3 improves translation initiation of mRNAs with N6-methyladenosine methylation at 5' untranslated region by facilitating eIF2AK2 recruitment in oxaliplatin-resistant colorectal cancer - RNA-seq cell line comparison
HCT116 and HCT8 cells were cultured within Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS). HCT116 cells were long-term cultured within DMEM containing 10% FBS and 1 μg/mL oxaliplatin. HCT8 cells were long-term cultured within DMEM containing 10% FBS and 0.2 μg/mL oxaliplatin. 1 x 107 cells were stored in 1 mL TRIZOL (Thermo Fisher Scientific) and ground in liquid nitrogen. Next, 100 μL chloroform was added, and the cells were fully mixed and centrifuged at the highest speed at 4 °C for 10 min. The supernatant was transferred into a new tube, isopropanol was added to the same volume, and cells were centrifuged at the highest speed at 4 °C for 10 min. The precipitate was washed with 75% cold ethanol and dissolved in DEPC water. The concentration and quality of RNA were measured using a Nanodrop 2000 (Thermo Fisher Scientific) and an Agilent Bioanalyzer 2100 (Agilent, USA). A total of 5 μg of RNA from each group was used for library preparation using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, USA) following the manufacturer's instructions