GSM5463937: Gastric adenocarcinoma cells - AGS NAT10 KO-2 Input; Homo sapiens; RIP-Seq
Total RNA was extracted from NAT10 knockout or control AGS cells using Trizol reagent, then digested with DNase I and fragmented into 100~200 nt nucleotides by RNA fragmentation reagent (Ambion). After saving 50ng fragmented RNA as input, the rest (150 μg) was used for RNA immunoprecipitation with ac4C antibody (Abcam). ac4C RNAs were immunoprecipitated with Dynabeads Protein G (Invitrogen), and purified with HiPure cell miRNA (Magen). Ribosomal RNA was removed from input RNA and ac4C-enriched RNA samples. RNA sequencing libraries for input RNA (RNA-seq) and ac4C-enriched RNA (acRIP-seq) were simultaneously constructed with the EpiTM mini longRNA-seq kit (Epibiotek), and then sequenced on the Illumina NovaSeq 6000 with two independent biological replicates. RNA libraries were prepared for sequencing using standard Illumina6000 protocols acRIP-seq