GSM8437027: RIP; Mus musculus; RIP-Seq
Cells were harvested in a lysis buffer(100 mM KCl, 5 mM MgCl2, 10 mM HEPES (pH 7.0), 0.5% NP40, 1 mM DTT, 100 units/mL RNaseOut) that was made in DEPC H2O. 10% of cell lysate was used for input. The left cell lysate was immunoprecipitated with 100 μL anti-FLAG M2 magnetic beads (M8823, Millipore) at 4 ℃ for 2 hours. The immunoprecipitated samples were washed using lysis buffer for 4 times. Total RNA was extracted from the immunoprecipitated and input samples using the TriPure Isolation Reagent (Roche, Mannheim, Germany). Message RNA was purified by using  Hieff NGS® mRNA Isolation Master Kit (12603, Yeasen Biotechnology). The fragment distribution and concentration of RNAs（after immunoprecipitation） were detected using an Agilent 2100 bioanalyzer (Agilent) and simpliNano spectrophotometer (GE Healthcare). Then, immunoprecipitated RNA or Input was used for library construction with NEB NextR Ultra™ RNA Library Prep Kit (New England Biolabs). Library quality was assessed on the Agilent Bioanalyzer 2100 system. The libraries were sequenced on an Illumina Novaseq 6000 platform with a paired-end read length of 150 bp according to the standard protocols.