VSV_wt_miCLIP-seq_rep2
mRNAs were purified using Dynabeads mRNA Purification Kit (Life Technologies, 61006) and fragmented to size around of 100 nt using the fragmentation reagent (Life Technologies, AM8740). 10 g of purified mRNAs were mixed with 25 g of anti-m6A antibody (Abcam, ab151230) in 450 l immunoprecipitation buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 0.05% NP-40) and incubated by rotating at 4 C for 2 h. The solution was then transferred to a clear flat-bottom 96-well plate (Corning) on ice and irradiated three times with 0.15 J/cm2 at 254 nm in a CL-1000 Ultraviolet Crosslinker (UVP). The mixture was then immunoprecipitated by incubation with Dynabeads Protein A (Life Technologies, 1001D) at 4 C for 2 h. After extensive washing and on-bead end-repair and linker ligation, the bound RNA fragments were eluted from the beads by proteinase K digestion at 55 C for 1 h. RNAs were isolated by further phenol-chloroform extraction and ethanol precipitation. Purified RNAs were used to construct the library using SMARTer smRNA-Seq Kit for Illumina (Takara, 635029) according to the manufacturers instructions. Sequencing was carried out on Illumina HiSeq X-ten platform with paired-end 150 bp read length.