GSM8105607: HeLa cells, SFTSV infected, Input2; Homo sapiens; OTHER
Total RNA was extracted using the Trizol method (Thermo Fisher Scientific, 15596018). DNase I (Roche Diagnostics, 04716728001) treatment was performed on the total RNA to remove residual DNA. Oligo-dT was used for mRNA enrichment.RNA was fragmented using RNA Fragmentation Buffer (100 mM Tris-HCl, 100 mM ZnCl2). The reaction was terminated with EDTA, and fragmented RNA was recovered. A portion of the fragmented RNA was used as input, while the remainder was used for immunoprecipitation (IP). The IP mixture was prepared using the following components: fragmented RNA, RNasin (40 U μl–1, Promega, cat. no. N2611), RVC (200 mM, Sigma-Aldrich, cat. no. R3380), IP buffer (50 mM Tris-HCl, 750 mM NaCl, 0.5% (vol/vol) IGEPAL CA-630), and m6A-specific antibody. The mixture was incubated at 4°C for 2 hours with rotation. Protein A beads (Thermo Fisher Scientific, 10002D) were washed twice with IP buffer, then suspended in IP buffer containing BSA (0.5 mg ml-1) and incubated with rotation for 2 hours at 4°C. The beads were washed twice with IP buffer. The IP mixture and the washed protein A beads were combined and incubated at 4°C for 2 hours with rotation, followed by three washes with IP buffer. The IP-eluted RNA was then purified using RNeasy Mini Kit (QIAGEN, 74106) RLT buffer. The Input RNA and IP RNA were subjected to library preparation using the NEBNext® Ultra™ II RNA Library Prep Kit (NEB E7770). Me RIP-seq