GSM7727533: mESC, Flag-MBD5, Input, rep1; Mus musculus; RIP-Seq
Pellets were lysed by rotating at 4 °C for 15 minutes after passing through a 26 G needle (BD Biosciences). Embryo suspensions were sonicated on a bioruptor (Diagenode) with 30 s on/30 s off for 5 cycles. Lysates were cleared by centrifugation at 21,000 g for 15 minutes at 4 °C on a benchtop centrifuge. Supernatants were applied to MBD6 antibody (LSbio, LS-C158106-400) conjugated protein A beads (Invitrogen, 1001D) and left overnight at 4 °C on an end-to-end rotor. Beads were washed extensively with 1 ml wash buffer (50 mM HEPES pH 7.5, 300 mM KCl, 0.05% (v/v) NP-40, 1 × Halt™ Protease and Phosphatase Inhibitor Cocktail, 1 × RNaseOUT Recombinant Ribonuclease Inhibitor) at 4 °C for 5 times. Protein-RNA complex conjugated to the beads were treated by 8 U/μL RNase T1 (Thermo Scientific, EN0541) at 22 °C for 10 minutes with shaking. Input samples are digested in parallel. Then input and IP samples were separated on an SDS-PAGE gel and gel slices at corresponding size ranges were treated by proteinase K (Invitrogen, 25530049) elution. RNA was recovered with TRIZol reagent (Invitrogen, 15596026). Then T4 PNK (Thermo Scientific, EK0031) end repair was performed. Library constructions were performed with NEBNext® Small RNA Library Prep Set for Illumina® (NEB, E7330S)