GSM8408762: acRIP-seq NAT10 knock out input in K562 cell line [KO_input_1]; Homo sapiens; RIP-Seq
Total RNA was rRNA depleted with the NEBNext® rRNA Depletion Kit v2 (Promega) and then subjected to immunoprecipitation. Briefly, RNA was randomly fragmented to ~100 nt by RNA Fragmentation Reagents. Protein A/G beads were coupled to the ac4C antibody or Normal Rabbit IgG antibody by rotating at room temperature for 2 h. The RNA fragments were incubated with the bead-linked antibodies and rotated at 4°C for 4 h. The RNA/antibody complexes are then digested with Proteinase K and the eluted RNA is purified by phenol:chloroform extraction. RNA extracted from IP, IgG and input samples obtained from NAT10 knock-out k562 cells was then constructed using the NEBNext® Ultra™ RNA Library Prep Kit,, following the manufacturer's instructions. Libraries were qualified using Agilent 2100 bioanalyzer and then sequenced in a Novaseq 6000 platform (Illumina).