RNA-Seq and m6A-RNA Immunoprecipitation (MeRIP-seq) Seq

RNA-seq was accomplished as previously reported (28). Total RNA was isolated from cells with TRIzol Reagent (Invitrogen). 1μg RNA was used to construct libraries with TruSeq Stranded total RNA Library Prep Kit (Illumina, San Diego, CA) and the residual RNA was used for RNA-seq. Sequencing was carried out on Illumina HiSeq 4000 according to the manufacturer’s instructions with single-end 50 bp read length. MeRIP-seq was accomplished as previously reported (31). Ribosomal RNA depleted RNA was isolated, purified by using RiboMinus™ Eukaryote Kit (Invitrogen, #A10837-08) and chemically shredded into ~100 nt fragments by using RNA fragmentation reagents (Invitrogen, #AM8740). RNA fragments (2000 ng) were denatured at 95°C for 3 min and incubated with 20 µl of Magna ChIP Protein A+G Magnetic Beads (Millipore, #2923270) conjugated to anti−m⁶A antibody (2.5 µg, Synaptic Systems, # 202003) or rabbit control IgG (Cell Signaling Technology) in 1X IPP buffer (15 mM NaCl, 10 mM Tris-HCl and 0.1% NP-40) with rotation at 4°C for 4 h. The beads were washed twice with 1X IPP buffer, twice with low−salt buffer (50 mM NaCl, 10 mM Tris-HCl and 0.1% NP-40), twice with high-salt buffer (500 mM NaCl, 10 mM Tris-HCl and 0.1% NP-40) and once with 1X IPP buffer. RNA was eluted from the beads with RLT buffer and purified through Qiagen RNeasy columns (Qiagen, #74104) according to the manufacturer’s recommendation. RNA fragments were purified from the eluates with RNA Clean and Concentrator (Zymo) and used to construct libraries with TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA). Sequencing was carried out on Illumina HiSeq 4000 according to the manufacturer’s instructions with single-end 50 bp read length.