GSM5203018: D2wt-input; Mus musculus; RIP-Seq
Total RNA was extracted from cells and organization using Trizol (Invitrogen) following the manufacturer's instructions. mRNA was first purified from total RNA using PolyATtract mRNA Isolation System IV (Promega Z5310). 2μg of the puriﬁed mRNA was fragmentized with ZnCl2 buffer at 94°C for 5min. The mRNA fragments were puriﬁed and then subjected to immunoprecipitation with α-m6A antibody (1:100, Synaptic Systems, Cat No: 202003). After extensive wash, the methylated fragments were eluted by competition using free N6-Methyladenosine (Santa Cruz Biotechnology, sc-215524) and then used for library construction with the TruSeq Stranded mRNA Sample Prep Kits (Illumina RS-122-2101). The input and m6A MeRIP libraries were sequenced with Illumina NextSeq 500 with high output kit. Libraries were prepared according to Illumina's instructions accompanying the TrueSeq Stranded mRNA library preparation kit.