GSM5516998: HPH2 input; Rattus norvegicus; RIP-Seq
The mRNA was isolated from total RNA with oligo(dT) magnetic beads (ThermoFisher), and then subjected to immunoprecipitation with the GenSeqTM m7G-IP Kit (GenSeq Inc., China) by following the manufacturer's instructions. Briefly, RNA was decapping with Tobacco Decapping Enzyme, and then randomly fragmented to ~200 nt by RNA Fragmentation Reagents. Protein A/G beads were coupled to the m7G antibody by rotating at room temperature for 1 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 libraries for IP and input samples were then constructed with NEBNext® Ultra II Directional RNA Library Prep Kit (New England Biolabs, Inc., USA) by following the manufacturer's instructions. Libraries were qualified using Agilent 2100 bioanalyzer and then sequenced in a HiSeq platform (Illumina).