GSM5776175: RA5_IP; Homo sapiens; OTHER
Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity was assessed by Bioanalyzer 2100 (Agilent, CA, USA) with RIN number >7.0. Total RNA  was used to deplete ribosomal RNA according to the manuscript of the Epicentre Ribo-Zero Gold Kit (Illumina, San Diego, USA).The poly(A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 86℃ 7min. Then the cleaved RNA fragments were incubated for 2h at 4℃ with m6A-specific antibody (No. 202003, Synaptic Systems, Germany) in IP buffer (50 mM Tris-HCl, 750 mM NaCl and 0.5% Igepal CA-630). The IP RNA was reverse-transcribed to create the final cDNA library in accordance with the protocol for the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, cat.e7530L, USA).