GSM5051629: Mettl3-WT-IN-CMP-1; Mus musculus; RNA-Seq
Total RNA was isolated using Tirzol reagent (Takara). Anti-m6A antibody (0.5 μg per sample) (ab15320, Abcam) was pre-bound to Protein A magnetic beads (Thermo Fisher Scientific) in IP buffer (10mM Tris-HCl pH 7.4, 100mM NaCl,0.1% NP40, , 0.4 U/μl RNasin) with head-to-tail mixing at 4 °C for 2 h. Total RNA (100 ng per sample) was added and incubated at 4°C for another 2 h. Beads were washed twice with 200 μl IP buffer, twice with 200 μl low-salt buffer (10mM Tris-HCl pH 7.4, 50 mM NaCl, 0.1% NP-40, 0.4 U/μl RNasin), twice with 200 μl high-salt buffer (10mM Tris-HCl pH 7.4, 500mM NaCl, 0.1% NP40, 0.4 U/μl RNasin) and once with 200 μl 1× IP buffer. RNA libraries were prepared for sequencing using standard Illumina protocols