GSM2878472: Flag_meRIP_2; Homo sapiens; RIP-Seq
mRNAs were extracted using GenElute™ mRNA Miniprep Kit (source: sigma, cat# MRN10) and were fragmented into 100nt length by using RNA Fragmentation buffer (0.1M Tris-HCL PH 7.0, 0.1M ZnCl2). 7ug Fragmented mRNAs were incubated for 2h at 4°C with 12.5ug of anti-m6A antibody (sysy, 202003) in IP buffer (0.05M Tris-HCL pH 7.4, 0.375M NaCl, 0.5% Igepal CA-630). The mixture was then subjected to immunoprecipitation by incubation with Pierce™ ChIP-grade Protein A/G Magnetic Beads (Thermo, 26162) at 4°C for 2h. After sufficient washing, bound RNA was eluted from the beads with Elution buffer (1×IP buffer, 7mM m6A, RNase inhibitor), and ethanol precipitated. The eluted RNA was resuspended in H2O and used to generate the cDNA library according to RNA-Seq Library Preparation Kit for Transcriptome Discovery–Illumina Compatible, which was then sequenced using the HiSeq 2000 system (Illumina) according to the manufacturer's instructions.