GSM7676912: KOG-Input for acRIP-seq; Homo sapiens; RIP-Seq
Total RNA was extracted using TRIzol™ Reagent (Invitrogen, 15596018). The concentration of total RNA was measured by Qubit RNA HS assay kit (Invitrogen, Q32852). 100μg 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). Acetylated RNA immunoprecipitation was performed using EpiTM ac4C immunoprecipitation kit (Epibiotek, R1815). Briefly, the fragmented RNA was incubated with Anti-N4-acetylcytidine (ac4C) antibody (abcam, ab252215) for 3 h at 4℃ and then with protein A/G magnetic beads (Thermo Fisher Scientific, 88802) at 4 ℃ for an additional 2 h to obtain immunoprecipitated RNA fragments. The ac4C-enriched RNA was purified by Zymo RNA clean and concentrator-25 kit (Zymo Research, R1017). The library was prepared by EpiTM mini longRNA-seq kit (Epibiotek, E1802). Both the input samples without IP and the ac4C IP samples were subjected to 150-bp, paired-end sequencing on an Illumina NovaSeq 6000 sequencer.