GSM8996603: MeRIP-seq IP, NC with LPS treatment Replicate 1; Mus musculus; OTHER
Total RNA was extracted using TRIzol™ Reagent (Invitrogen, 15596018). RNA-seq: VAHTS Stranded mRNA-seq Library Prep Kit for Illumina V2 (Vazyme Biotech, NR612-02) was used for library preparation according to the instructions. MeRIP-seq: 10 μg total RNA was fragmented into 100-200 nt RNA fragments using 10X RNA Fragmentation Buffer (100 mM Tris-HCl, 100 mM ZnCl2 in nuclease-free H2O). The reaction was stopped by adding 10X EDTA (0.5M EDTA). Methylated RNA immunoprecipitation was performed using EpiTM m6A immunoprecipitation kit (Epibiotek, R1804). Briefly, the fragmented RNA was incubated with anti-m6A monoclonal antibody (abcam,ab208577) for 3 h at 4℃ and then with protein A/G magnetic beads (Invitrogen, 8880210002D/10004D) at 4 ℃ for an additional 2 h to obtain immunoprecipitated RNA fragments. The m6A-enriched RNA was purified using TRIzol™ Reagent (Invitrogen, 15596018). The library was prepared by smart-seq method. Ribo-seq: A total of 10^7 cells were prepared for cell lysis. Then, the cell medium was discarded, and the cells were washed twice with ice-cold PBS containing 100 μg/mL cycloheximide (Adooq, 66-81-9). Ribosome profiling was performed using Epi™ Ribosome Profiling Kit (Epibiotek, R1814). Subsquently, RPFs (ribosome-protected RNA fragments) was extracted using RNA clean&ConcentratorTM-5 kit (ZYMO, R1016). EpiTM RiboRNA Depletion Kit (Human/Mouse/Rat) (Epibiotek, R1805) was used for rRNA depletion. Sequencing libraries were constructed using QIAseq miRNA Library kit (QIAGEN, 1103679).