GSM4565840: m6A_IP_WT_rep2; Mus musculus; RIP-Seq
Total RNA for RNA-seq was extracted with Trizol and the Direct-zol RNA Kit. For m6A seq, polyA RNAs were enriched with the Dynabeads mRNA Purification Kit and fragmented to 150 nt with Bioruptor Pico sonicator. Fragments containing m6A were enriched with the EpiMark N6-methyladenosine Enrichment Kit. To perform RIP-seq, YTHDF2 antibodies were incubated with Protein A Dynabeads. Cells were lysed with lysis buffer (50 mM HEPES, 150 mM KCl, 2 mM EDTA, 0.5% NP40, 0.5 mM DTT, protease and RNase inhibitors). Debris was removed and the lysate was incubated with the protein-bound-beads at 4C for 4 hours. The beads were washed with wash buffer (50 mM HEPES, 200 mM NaCl, 2 mM EDTA, 0.05% NP40, 0.5 mM DTT, protease and RNase inhibitors). YTHDF2 bound RNAs were eluted with Trizol. All libraries were constructed with the SMARTer Stranded Total RNA-Seq Kit v2 (Takara)
GEO Accession
GSM4565840