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 - RIP-seq dataset
HCT116 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. 1×107 cells were harvested, resuspended in nuclear isolation buffer (1.28 M sucrose, 40 mM Tris pH 7.5, 20 mM MgCl2, 4% Triton X-100) and kept on ice for at least 30 min with frequent mixing. The pellet nuclei were centrifuged with 14000 rpm for 15 min, resuspended by wash buffer (150 mM KCl, 25 mM Tris pH 7.4, 5 mM EDTA, 0.5 mM DTT, 0.5% NP40, 100 U/mL RNAase inhibitor (Solarbio, Beijing, China), 1×Protease inhibitors cocktail (Solarbio) and sheared the chromatin through sonication by high power, 5 s on, 30 s off for 30 cycles. After that, 90% nuclei were incubated with 1 μg antibodies of m6A methylation (Cat No. 56593S, CST, Beverly, USA), YTHDF3, eIF3A (Cat No. NBP1-18891, NOVUS, Centennial, USA) or eIF2AK2 overnight and 40 μL Protein A/G beads (Thermo Fisher Scientific) 2 h by gentle rotation at 4 °C while the rest of 10% were harvested as input. The pellet beads were centrifuged by 3000 rpm 3 min, washed three times. Both the input and pellet beads were purified by RNAiso plus (Takara, Kusatsu, Japan) and conducted reverse transcription using QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). Libraries of cDNA were used for next generation sequencing or qPCR. For sequencing, the concentration and quality of purified RIP RNAs was measured by Nanodrop 2000 (Thermo Fisher Scientific) and Agilent bioanalyzer 2100 (Agilent, Santa Clara, USA). 4 μg RNA in each group were used for library preparation by NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB) following manufacturer's instructions