GSM5462332: YO_P3; Bos grunniens; RIP-Seq
The total RNA was isolated and purified using the TRIzol reagent . The amount and purity of the RNA samples were quantified using the NanoDrop ND-1000. The Poly (A) RNA was purified from 50 μg total RNA using Dynabeads Oligo (dT) 25-61005through two rounds of purification. Then the captured RNA was fragmented into small pieces using the Magnesium RNA Fragmentation Module for 7 min at 86°C. The fragmented RNA was premixed with the Dynabeads Antibody Coupling Kit and m6A antibody（No.202003, Synaptic Systems, Germany）  in IP buffer (50 mM Tris-HCl, 750 mM NaCl and 0.5% Igepal CA-630) and incubated for 2 h at 4°C. The IP RNA was reverse-transcribed to synthesize the cDNA using the SuperScript™ II Reverse Transcriptase. Then the two-strand synthesis was performed with RNase H using the E. coli DNA polymerase I  to synthesize U-labeled second-stranded DNAs. At the same time, the dUTP Solution was incorporated into the two strands, and the ends of the double-stranded DNA were filled with blunt ends. An A-base was added at each end of the fragment to connect it to the T-base overhang, and the size of the fragment was screened and purified using the AMPureXP beads. The U-labeled second-stranded DNAs were digested using a heat-labile UDG enzyme , then amplified by PCR under the following conditions: Pre-denaturation at 95°C for 3 min, denaturation at 98°C (8 cycles) for 15 s, annealing at 60°C for 15 s, extending at 72°C for 30 s, and finally extending at 72°C for 5 min to form a cDNA library of 300 ± 50 BP. At last, the 2 × 150 bp paired-end sequencing (PE150) was performed on an Illumina Novaseq™ 6000.