GSM5961633: HEK293T_input; Homo sapiens; RIP-Seq
Total RNA was isolated from tissues using chloroform and isopropanol following the manufacturer's protocol. Polyadenylated RNA was separated from total RNA using Oligo dT Magnetic Beads (Vazyme). RNA samples were fragmented in 1X NEB Next Magnesium RNA Fragmentation Buffer at 94℃ for 5min and fragmented RNA was then cleaned up using ethanol precipitation. 10ng fragmented RNA was used to construct input control library with VAHTS stranded mRNA-seq library prep kit (Vazyme). 15-40ug fragmented RNA was further incubated with 5ug rabbit anti-m6A polyclonal antibody (Synaptic Systems, catalog number 202003) in IPP buffer overnight at 4℃. The m6A-Ab mixture was then immunoprecipitated by incubation with protein-G magnetic beads at 4℃ for another 2h. After washing with IPP buffer, bound RNA was competitively eluted from the beads with 0.5mgml-1 N6-methyladenosine (Sigma-Aldrich), followed by ethanol precipitation. RNA was resuspended in 8μl water and used for library construction.