GSM3462689: HEK293T_input (RIP-seq); Homo sapiens; RIP-Seq
Total RNA was isolated from cultured cells using chloroform and isopropanol following the manufacturer's protocol. Polyadenylated RNA was separated from total RNA using Oligo dT Magnetic Beads (Vazyme). Polyadenylated mRNA was first fragmented with Magnesium RNA Fragmentation Module and then cleaned up using ethanol precipitation. 100ng RNA was saved to serve as input control and the rest was incubated with 5ug anti-m5C antibody in IPP buffer overnight at 4℃. 60μl Protein-G magnetic beads was pre-blocked with 0.5mgml-1 BSA at 4℃ for 2h and then added for immunoprecipitating m5C-Ab mixture at 4℃ for another 2h. After washing with IPP buffer 3 times, RNA was competitively eluted from the beads with 20mM 5-Methylcytosine hydrochloride , followed by ethanol precipitation. RNA was resuspended in 8μl water. VAHTS Stranded mRNA-seq library prep kit (Vazyme) was used for library construction of both IP and input RNAs.