GSM4859987: WT-rep1-Inp; Mus musculus; RIP-Seq
Total RNA was extracted from the mESCs using TRIzol  reagent. Enrichment of mRNA from total RNA was performed using a Dynabeads mRNA  Purification Kit (Invitrogen), according to the manufacturer's instructions. mRNA samples were  chemically fragmented into ~100-nucleotide-long fragments by incubation with 10X RNA  Fragmentation Reagent (Invitrogen) at 90°C for 90 seconds. The fragmentation reaction was  stopped by adding 0.5 M EDTA, followed by standard ethanol precipitation. The fragmented RNA  samples were resuspended in 10 mM Tris-HCl (pH 7.4). 20 ng of fragmented RNA to be used as  input control was stored in -80°C in a final volume of 8.5 μl in FPF (Fragment, Prime, Finish) mix  from the TruSeq Stranded Total RNA Kit (Illumina). The remaining RNA was subjected to m6Aseq.  Specifically, 2 μg of fragmented mRNA was incubated for 2 h at 4°C with 4 μg of affinitypurified  anti-m6A polyclonal antibody (Synaptic Systems) in m6A-IP buffer (150 mM NaCl, 0.1%  NP-40, 10 mM Tris-HCl, [pH 7.4], and 0.2 U/μl of RNasin). The RNA–antibody mixture was  immunoprecipitated by incubation with protein A beads (Millipore) at 4°C for another 2 h. The  beads were extensively washed with m6A-IP buffer. The bound RNA was eluted with m6A elution  buffer (150 mM NaCl, 6.7 mM m6A nucleotides, 0.1% NP-40, 10 mM Tris-HCl, [pH 7.4], and 0.2  U/μl of RNasin) and elution wash buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, [pH 7.4],  0.2 U/μl of RNasin). The m6A eluate was purified using Agencourt AMPure XP beads (Beckman  Coulter). The immunopurified RNA and non-IP input control samples were used for library  generation using the TruSeq Stranded Total RNA Kit (Illumina). library strategy: MeRIP-seq