title: GSM5696542: OE_2_IP; Homo sapiens; RIP-Seq
library_construction_protocol: ~5-20 × 106 cells were harvested and lysed. Protein A/G MagBeads pre-coated with 5μg of the antibody of FLAG and incubated with cell lysate supernatant overnight at 4°C.The beads containing immunoprecipitated RNA-protein complex were treated with 150μL of Proteinase K buffer to digest the proteins. Specific binding RNAs were isolated by using a phenol-chloroform lysate to obtain a purified product. RNA samples were fragmented by RNA fragmentation buffer (10 mM Tris-HCl [pH 7.0], 10 mM ZnCl2). Fragmented RNA was incubated for 2 hr at 4C with 0.5 mg/mL antim6A polyclonal antibody in IP buffer (150 mM NaCl, 0.1% Igepal CA-630, 10 mM Tris-HCl [pH 7.4]) supplemented with RNasin Plus RNase inhibitor (Promega). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase（RNase H - ）. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µL USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Illumina HiSeqTM 2500 system.