GSM5004427: ES rep-1; Mus musculus; RIP-Seq
The cell pellets were resuspended in RIP buffer. Then treated the cell lysate with DNase I. The pre-equilibrated FLAG M2 resins were incubated with the lysate at 4°C overnight. The RNA/Protein complexes were eluted with 3 × FLAG elution buffer, then, incubated with pre-equilibrated streptavidin beads at 4°C overnight. Then the RNAs were partially digested with Micrococcal nuclease at 37°C for 10 min. The RNAs were purified by TRIzol™ Reagent and ligated with 5′ RNA adaptor. the purified RNA fragments were reverse transcribed by Superscript III (Invitrogen) with RT primer and amplified for 20 cycles with 2× NEB HF PCR mix (NEB). Library index sequence was introduced by PCR with index primers for eight more cycles. Library was performed high-throughput sequencing by Illumina Hiseq 2500 sequencing platform.