Bioruptor plus sonicator device

Polyadenylated RNAs from Zc3h13 knockdown and control mESCs were prepared as described above, and sonicated to 100–200 nt fragments by Bioruptor Plus sonicator device (Diagenode). A small portion (10%) of the RNA fragments was left aside to be used as input sample. MeRIP was performed as previously described with minor modifications (Dominissini et al., 2012 (link)). Briefly, 4 μg fragmented polyadenylated RNA was incubated with 2 μg anti-m⁶A antibody (Synaptic Systems) in 1 x IP buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% NP-40) for 2 hr at 4°C. The m⁶A-IP mixture was then incubated with Dynabeads protein A (Life Technologies) for an additional 2 hr at 4°C on a rotating wheel. After washing 3 times with 1 x IP buffer, the bound RNA was eluted by competition with N⁶-methyladenosine (Santa Cruz Biotechnology) and then purified using an RNA cleanup kit (Zymo Research). The purified RNA fragments from MeRIP were used for library construction using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, Cat#E7420) following manufacturer’s instructions and sequenced with Illumina HiSeq 2000 or Illumina HiSeq X10.
For MeRIP-seq of cytoplasmic and nuclear fraction samples, RNAs were extracted from cytoplasmic or nuclear fractions of Zc3h13 kd mESCs and control cells. MeRIP was then performed according to the same procedure above.

For chromatin RNA MeRIP-seq, chromatin fraction was collected as above. RNAs in chromatin fraction were isolated using TRIzol reagent (Invitrogen, #15596018). 20 μg RNA was sonicated to 100–200nt fragments by Bioruptor Plus sonicator device (Diagenode) and incubated with 5μg anti-m⁶A antibody in 1 × IP buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% NP-40) for 2 hours at 4°C. The m⁶A-IP mixture was then incubated with Dynabeads protein A/G for an additional 2 hours at 4°C on a rotating wheel. After washing 3 times with 1 × IP buffer, the bound RNA was isolated with TRIzol reagent. The purified RNA fragments from MeRIP and Input RNA were first treated with Ribo-off rRNA Depletion Kit (Vazyme, #N406), followed with library construction using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, #E7420) according to manufacturer’s guidelines.