GSM4889072: round spermatids_m6A input_rep2; Mus musculus; RIP-Seq
total RNA was harvested using Trizol reagent. PolyA mRNA was enriched using GenElute mRNA Miniprep Kit. mRNA was sonicated to ~100 nt, mixed with 2.5 mg affinity-purified anti-m6A polyclonal antibody (Synaptic Systems) in IP buffer (150 mM NaCl, 0.1% NP-40, and 10 mM Tris-HCl, pH 7.4), and incubated for 2 h at 4 °C. The an- tibody-RNA complex was isolated by incubation with protein A beads (Invitrogen) at 4 °C for 2 h. The beads were washed three times and eluted competitively with an m6A monophosphate solution. RNA in the eluate was isolated using RNA Clean and Concentrator (Zymo Research) and used for library preparation with TruSeq stranded mRNA sample preparation kit (Illumina). RNA in the eluate was isolated using RNA Clean and Concentrator (Zymo Research) and used for library preparation with TruSeq stranded mRNA sample preparation kit (Illumina).