GSM8241016: LargeWhite pig muscle, replicate1, 5-day-old, MeRIP-seq; Sus scrofa; RIP-Seq
Total RNA was extracted using Trizol reagent (Thermo Fisher Scientific) and the concentration of total RNA were measured by Qubit RNA HS Assay. mRNA was isolated from 75 ug of total RNA using Dynabeads™ mRNA Purification Kit (Thermo Fisher Scientific). The purified mRNA was then fragmented in 1X RNA Fragmentation Buffer (Thermo Fisher Scientific) at 70℃ for 5 min. After purification, the fragmented RNA was used for enrichment with 5 ug of m6A antibody (Synaptic Systems) which were previously incubated with 25ul of protein A/G magnetic beads mixture (Thermo Fisher Scientific). The incubation was performed at 4℃ for 4h with rotation. After immunoprecipitation, the magnetic beads were washed 3 times using IP buffer (150 mM NaCl, 10 mM Tris-HCl pH 7.5, 0.1% IGEPAL CA-630 in nuclease-free H2O) for 10 min of each time at 4℃ with rotation. Finally, the m6A modified mRNA fragments were extracted by Trizol reagent and purified with RNA Clean & Concentrator™ Kit (Zymo Research) After purification, both m6A antibody enriched RNA and partial of unenriched mRNA after fragmentation which was used as input was constructed library using VAHTS® mRNA-seq V3 Library Prep Kit for Illumina(Vazyme) according to the manufacturer's protocol. MeRIP-seq