Instrument: HiSeq X Ten
Strategy: RIP-Seq
Source: TRANSCRIPTOMIC
Selection: other
Layout: PAIRED
Construction protocol: Total RNA was extracted using TRIzol Reagent (Ambion) following the manufacturer's instructions, and mRNA were then purified by GenElute™ mRNA Miniprep Kit (sigma, MRN10). The polyadenylated mRNAs isolated from total RNA using GenElute™ mRNA Miniprep Kit (sigma, MRN10) were fragmented into 100 nt length by using RNA Fragmentation buffer (0.1M Tris-HCL PH 7.0, 0.1M ZnCl2). Then, 50 and 100 ng fragmented mRNAs were incubated for 2 hours at 4 °C with 12.5 μg 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 2 hours. After sufficient washing, m6A antibody-bound RNA was eluted from the beads with Elution buffer (1×IP buffer, 7mM m6A, RNase inhibitor), and then 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.